Non-human animals expressing exogenous terminal deoxynucleotidyltransferase

ABSTRACT

Provided herein are methods and compositions related to non-human animals that express exogenous Terminal Deoxynucleotidyltransferase (TdT).

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/345,524, filed Jun. 3, 2016, herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 12, 2017, isnamed RPB-013.01_SL.txt and is 7,930 bytes in size.

BACKGROUND

Non-human animals, particularly mice and rats, have proven to be avaluable source of therapeutic antibodies and potentially could serve asa source of other antigen binding molecules. A high level of antigenreceptor diversity in such non-human animals increases the likelihoodthat an antigen binding molecule having desirable therapeutic propertieswill be generated following immunization. Accordingly, there is a needfor genetically engineered non-human animals that have increased antigenreceptor diversity to improve production of therapeutic antigen bindingmolecules.

SUMMARY

In certain aspects, provided herein are genetically modified non-humananimals comprising in their genome an exogenous nucleic acid encodingterminal deoxynucleotidyltransferase (TdT), as well as methods of makingand using such non-human animals. In some embodiments, the exogenous TdTis human TdT. In some embodiments, the exogenous TdT is of endogenousspecies origin (e.g., in mice the exogenous TdT has a mouse sequence).In some embodiments, the non-human animals provided herein express theTdT encoded by the exogenous nucleic acid during B cell development, forexample, in pro-B cells and/or in pre-B cells. In some embodiments, thenon-human animals provided herein express the TdT encoded by theexogenous nucleic acid during T cell development, for example, indouble-negative (DN) thymocytes and/or in double-positive (DP)thymocytes. In some embodiments, the genetically modified non-humananimal comprises multiple copies of exogenous nucleic acids encoding TdT(e.g., at least 2, 3, 4, 5, 6, 7 or 8 copies). In some embodiments, thegenetically modified non-human animal is a mammal, such as a rodent(e.g., a mouse or a rat).

In some embodiments, the genetically modified non-human animal comprisesin its genome an immunoglobulin variable region comprising unrearrangedhuman immunoglobulin variable region gene segments (e.g., heavy chaingene segments, κ chain gene segments, λ chain gene segments) operablylinked to an immunoglobulin constant region gene (e.g., a heavy chainconstant region gene, a κ chain constant region gene, a λ chain constantregion gene). In some embodiments, the constant region gene is a humanconstant region gene, a mouse constant region gene or a rat constantregion gene. In some embodiments, the constant region gene is ofendogenous species origin. In some embodiments, the variable region andthe constant region gene are located in an endogenous immunoglobulinlocus (e.g., a heavy chain locus, a κ locus, a λ locus). In someembodiments, the genetically modified non-human organism expressesantibodies comprising a human immunoglobulin variable domain derivedfrom the immunoglobulin variable region and an immunoglobulin constantdomain encoded by the immunoglobulin constant region gene. In someembodiments, provided herein are methods of using such a geneticallymodified non-human animal to generate an antibody, a B cell, a hybridomaor a nucleic acid encoding a human immunoglobulin variable domain.

In certain embodiments, the genetically modified non-human animalcomprises in its genome a T cell receptor (TCR) variable regioncomprising unrearranged human TCR variable region gene segments (e.g.,TCRα gene segments, TCR β gene segments, TCRγ gene segments, TCRδ genesegments) operably linked to a TCR constant region gene (e.g., TCRαconstant region gene, TCR β constant region gene, TCRγ constant regiongene, TCRδ constant region gene). In some embodiments, the constantregion gene is a human constant region gene, a mouse constant regiongene or a rat constant region gene. In some embodiments, the constantregion gene is of endogenous species origin. In some embodiments, thevariable region and the constant region gene are located in anendogenous TCR locus (e.g., TCRα locus, TCR β locus, TCRγ locus, TCRδlocus). In some embodiments, the genetically modified non-human organismexpresses TCR comprising a human TCR variable domain derived from theTCR variable region and a TCR constant domain encoded by the TCRconstant region gene. In some embodiments, provided herein are methodsof using such a genetically modified non-human animal to generate a TCR,a T cell, a T cell hybridoma or a nucleic acid encoding a human TCRvariable domain.

In some embodiments, the genetically modified non-human animal comprisesin its genome an immunoglobulin variable region comprising unrearrangedhuman immunoglobulin variable region gene segments (e.g., heavy chaingene segments, κ chain gene segments, λ chain gene segments) operablylinked to a TCR constant region gene (e.g., TCRα constant region gene,TCR β constant region gene, TCRγ constant region gene, TCRδ constantregion gene). In some embodiments, the constant region gene is a humanconstant region gene, a mouse constant region gene or a rat constantregion gene. In some embodiments, the constant region gene is ofendogenous species origin. In some embodiments, the variable region andthe constant region gene are located in an endogenous TCRδ locus (e.g.,TCRα locus, TCR β locus, TCRγ locus, TCRδ locus). In some embodiments,the genetically modified non-human organism expresses chimeric antigenreceptor (CAR) comprising a human immunoglobulin variable domain derivedfrom the immunoglobulin variable region and a TCR constant domainencoded by the TCR constant region gene. In some embodiments, providedherein are methods of using such a genetically modified non-human animalto generate an CAR, a T cell, a T cell hybridoma or a nucleic acidencoding a human immunoglobulin variable domain.

In some embodiments, provided herein are methods of making a non-humananimal disclosed herein comprising engineering the non-human animal tocomprise in its germline the genetic modifications described herein. Insome embodiments, provided herein are non-human ES cells comprising thegenetic modifications described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a diagram of an exemplary targeting vector (not to scale)whereas part of the mouse Rag2 gene is replaced with a DNA sequenceencoding short isoform human TdT (hTdTs). In exemplary embodiments, thevector is randomly integrated into the genome. Unless labeling in thediagram suggests otherwise (e.g., as for selection cassettes, loxPsites, etc.), filled shapes and single lines represent mouse sequences,and empty shapes and double lines represent human sequences. E1, E2,etc. represent exons of particular illustrated genes, GFP is greenfluorescent protein, CM is chloramphenicol resistant gene, neo isneomycin resistant gene. Junctions 1-4 correspond to junctions indicatedin Table 1.

FIG. 2 depicts a diagram of an exemplary targeting vector (not to scale)where a part of the mouse Rag2 gene is replaced with a DNA sequenceencoding short isoform human TdT (hTdTs). In the depicted embodiment,the vector is used to insert hTdT, driven by the mouse RAG2 promoter,into the Ig kappa locus. Unless labeling in the diagram suggestsotherwise (e.g., as for selection cassettes, loxP sites, etc.), filledshapes and single lines represent mouse sequences, and empty shapes anddouble lines represent human sequences. E1, E2, etc. represent exons ofparticular illustrated genes, GFP is green fluorescent protein, CM ischloramphenicol resistant gene, hyg is hygromycin resistant gene.Junctions 1-7 correspond to junctions in Table 2.

FIG. 3 depicts a diagram of an exemplary targeting vector (not to scale)used to insert a DNA sequence encoding human TdT (hTdTs), driven byVH1-72 promoter and Eμ enhancer, into the immunoglobulin κ locus. Unlesslabeling in the diagram suggests otherwise (e.g., as for selectioncassettes, loxP sites, etc.), filled shapes and single lines representmouse sequences, and empty shapes and double lines represent humansequences. E1, E2, etc. represent exons of particular illustrated genes,GFP is green fluorescent protein, CM is chloramphenicol resistant gene,hyg is hygromycin resistant gene. Junctions 1-4 correspond to junctionsin Table 3.

FIG. 4 depicts expression of hTdT mRNA in lymphocytes of VELOCIMMUNE®TdT mice compared to VELOCIMMUNE® control mice. VELOCIMMUNE® mice hereinare mice that comprise a diverse repertoire of unrearranged human heavychain and kappa light chain variable (V(D)J) gene segments. Hetindicates a heterozygous mouse, HO indicates a homozygous mouse.

FIG. 5 depicts a graph showing hIgκ sequence diversity (# of uniquelight chain CDR3 sequences per 10,000 hIgκ sequencing reads) inVELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice.Het indicates a heterozygous mouse, HO indicates a homozygous mouse.

FIG. 6 depicts a graph showing the distribution of hIgκ non-templateadditions in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE®control mice. Het indicates a heterozygous mouse, HO indicates ahomozygous mouse. “NT” stands for nucleotides.

FIG. 7 has two panels. Panel (A) depicts a graph showing thedistribution of hIgκ CDR3 lengths in VELOCIMMUNE® mice expressing hTdTcompared to VELOCIMMUNE® control mice. “AA” stands for amino acid. Panel(B) depicts a graph showing exonuclease deletion length frequencies at5′ region of JK segments in VELOCIMMUNE® mice expressing hTdT comparedto VELOCIMMUNE® control mice. Het indicates a heterozygous mouse, HOindicates a homozygous mouse.

FIG. 8 has two panels. Panel (A) depicts a graph showing Vκ usage inVELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice.Panel (B) depicts a graph showing J_(K) usage in VELOCIMMUNE® miceexpressing hTdT compared to VELOCIMMUNE® control mice. Het indicates aheterozygous mouse, HO indicates a homozygous mouse.

FIG. 9 depicts a graph showing mIgλ sequence diversity (# of uniquelight chain CDR3 sequences per 10,000 Igλ sequencing reads) inVELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice.Het indicates a heterozygous mouse, HO indicates a homozygous mouse.

FIG. 10 depicts a graph showing the distribution of mIgλ non-templateadditions in VELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE®control mice. Het indicates a heterozygous mouse, HO indicates ahomozygous mouse. “NT” stands for nucleotides.

FIG. 11 depicts a graph showing the distribution of mIgλ CDR3 lengths inVELOCIMMUNE® mice expressing hTdT compared to VELOCIMMUNE® control mice.Het indicates a heterozygous mouse, HO indicates a homozygous mouse.“AA” stands for amino acid.

FIG. 12 depicts a graph showing Vλ usage in VELOCIMMUNE® mice expressinghTdT compared to VELOCIMMUNE® control mice. Het indicates a heterozygousmouse, HO indicates a homozygous mouse.

FIG. 13 depicts a graph showing hIgλ sequence diversity (# of uniquelight chain CDR3 sequences per 10,000 I_(D)(sequencing reads) in duallight chain mice (DLC; mice comprising two unrearranged human Vk genesegments and five unrearranged human Jk gene segments, as well as adiverse repertoire of unrearranged human heavy chain V, D, and J genesegments) expressing hTdT (right panel; hTdT genes present as indicated)compared to VELOCIMMUNE® mice expressing hTdT (left panel; hTdT genespresent as indicated) and DLC and VELOCIMMUNE® control mice that do notexpress hTdT. Het indicates a heterozygous mouse for hTdT, HO indicatesa homozygous mouse for hTdT.

FIG. 14 depicts a graph showing the distribution of hIgλ non-templateadditions in mice expressing hTdT compared to DLC control mice notexpressing hTdT (DLC). Het indicates a heterozygous mouse for hTdT, HOindicates a homozygous mouse for hTdT. “NT” stands for nucleotides.

FIG. 15 depicts a graph showing the distribution of hIgκ CDR3 lengths inDLC mice expressing hTdT compared to DLC control mice not expressinghTdT. Het indicates a heterozygous mouse for hTdT, HO indicates ahomozygous mouse for hTdT.

FIG. 16 depicts graphs showing Vκ usage and Jκ usage in DLC miceexpressing hTdT compared to DLC control mice not expressing hTdT. Hetindicates a heterozygous mouse for hTdT, HO indicates a homozygous mousefor hTdT. Only two different Rag TdT tg (HO) DLC mice where used, whichare depicted separately.

DETAILED DESCRIPTION

General

Provided herein are methods and compositions related to non-humananimals comprising in their genome an exogenous nucleic acid encodingTdT (e.g., human, mouse or rat TdT). In some embodiments, thegenetically modified non-human animal is a mammal, such as a rodent(e.g., a mouse or a rat). In certain embodiments, the genome of thenon-human animal comprises further modifications such that it expressesantigen binding molecules having human variable domains (e.g.,antibodies, TCRs and/or CARs).

TdT is a DNA polymerase that catalyzes template-independent addition ofnucleotides (N-additions) during junction formation in V(D)Jrecombination, which leads to an increase in antigen-receptor diversityin B and T lymphocytes. In some embodiments, the non-human animalsprovided herein express increased levels of TdT during B celldevelopment and/or T cell development compared to correspondingnon-human animals (i.e., non-human animals of the same species andstrain) that do not include in their genome an exogenous nucleic acidencoding TdT. In some embodiments, the non-human animals provided hereinexpress TdT during stages of B cell development and/or T celldevelopment during which corresponding non-human animals that do notinclude in their genome an exogenous nucleic acid encoding TdT do notexpress TdT (e.g., during the pre-B cell stage). In some embodiments,the genetically modified non-human animals described herein haveincreased antigen-receptor diversity (e.g., antibody diversity, TCRdiversity and/or CAR diversity) compared to corresponding non-humananimals that do not include in their genome an exogenous nucleic acidencoding TdT.

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof amino acid analogs having variant side chains; and allstereoisomers of any of the foregoing.

As used herein, the term “antibody” may refer to both an intact antibodyand an antigen binding fragment thereof. Intact antibodies areglycoproteins that include at least two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chain includesa heavy chain variable region (abbreviated herein as V_(H)) and a heavychain constant region. Each light chain includes a light chain variableregion (abbreviated herein as V_(L)) and a light chain constant region.The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The term “antibody” also includes single domain antibodies,heavy chain only antibodies, antibodies with light chain variable genesegments on heavy chain, etc.

The terms “antigen binding fragment” and “antigen-binding portion” of anantigen binding molecule (e.g., an antibody, a T cell receptor (TCR), achimeric antigen receptor (CAR)), as used herein, refers to one or morefragments of the antigen binding molecule that retain the ability tobind to an antigen. An antigen binding fragment can include anyantibody, TCR or CAR fragment that retains at least a portion of thevariable region of an intact antigen binding molecule and is capable ofbinding to an antigen. Examples of binding fragments encompassed withinthe term “antigen binding fragment” include, but are not limited to Fab,Fab', F(ab')₂, Fv, scFv, disulfide linked Fv, Fd, single-chainantibodies, soluble TCR, single-chain TCR, soluble CAR, single-chainCAR. isolated CDRH3 (antibody or TCR), and other antigen bindingfragments that retain at least a portion of the variable region of anintact antigen binding molecule. These antigen binding fragments can beobtained using conventional recombinant and/or enzymatic techniques andcan be screened for antigen binding in the same manner as intactantibodies.

The term “corresponding” in reference to a non-human animal, is used todescribe the features of a control non-human animal of the same speciesand comprising the same genetic modifications as a subject non-humanexcept that the subject non-human animal expresses exogenous TdT whereasthe corresponding non-human animal does not.

As used herein, a “chimeric antigen receptor” or “CAR” refers to anantigen binding protein in that includes an immunoglobulin antigenbinding domain (e.g., an immunoglobulin variable domain) and a T cellreceptor (TCR) constant domain or a portion thereof. As used herein, a“constant domain” of a TCR polypeptide includes a membrane-proximal TCRconstant domain, and may also include a TCR transmembrane domain and/ora TCR cytoplasmic tail. For example, in some embodiments, the CAR is adimer that includes a first polypeptide comprising an immunoglobulinheavy chain variable domain linked to a TCRβ constant domain and asecond polypeptide comprising an immunoglobulin light chain variabledomain (e.g., a κ or λ variable domain) linked to a TCRα constantdomain. In some embodiments, the CAR is a dimer that includes a firstpolypeptide comprising an immunoglobulin heavy chain variable domainlinked to a TCRα constant domain and a second polypeptide comprising animmunoglobulin light chain variable domain (e.g., a κ or λ variabledomain) linked to a TCRβ constant domain.

The phrase “derived from” when used concerning a rearranged variableregion gene or a variable domain “derived from” an unrearranged variableregion and/or unrearranged variable region gene segments refers to theability to trace the sequence of the rearranged variable region gene orvariable domain back to a set of unrearranged variable region genesegments that were rearranged to form the rearranged variable regiongene that expresses the variable domain (accounting for, whereapplicable, splice differences and somatic mutations). For example, arearranged variable region gene that has undergone somatic mutation doesnot change the fact that it is derived from the unrearranged variableregion gene segments.

As used herein, the term “locus” refers to a region on a chromosome thatcontains a set of related genetic elements (e.g., genes, gene segments,regulatory elements). For example, an unrearranged immunoglobulin locusmay include immunoglobulin variable region gene segments, one or moreimmunoglobulin constant region genes and associated regulatory elements(e.g., promoters, enhancers, switch elements, etc.) that direct V(D)Jrecombination and immunoglobulin expression, while an unrearranged TCRlocus may include TCR variable region gene segments, a TCR constantregion gene and associated regulatory elements (e.g., promoters,enhancers, etc.) that direct V(D)J recombination and TCR expression.Similarly, an unrearranged CAR locus may include immunoglobulin variableregion gene segments, a TCR constant region gene and associatedregulatory elements (e.g., promoters, enhancers, etc.) that direct V(D)Jrecombination and CAR expression. A locus can be endogenous ornon-endogenous. The term “endogenous locus” refers to a location on achromosome at which a particular genetic element is naturally found.

Unrearranged variable region gene segments are “operably linked” to acontiguous constant region gene if the unrearranged variable region genesegments are capable of rearranging to form a rearranged variable regiongene that is expressed in conjunction with the constant region gene as apolypeptide chain of an antigen binding protein.

The terms “polynucleotide”, and “nucleic acid” are used interchangeably.They refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three-dimensional structure, and mayperform any function. The following are non-limiting examples ofpolynucleotides: coding or non-coding regions of a gene or genefragment, loci (locus) defined from linkage analysis, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. A polynucleotide may be furthermodified, such as by conjugation with a labeling component. In allnucleic acid sequences provided herein, U nucleotides areinterchangeable with T nucleotides.

As used herein, “specific binding” and “antigen specificity” refers tothe ability of an antigen binding molecule (e.g., an antibody, a TCR, aCAR) to bind to a predetermined target, such as a predetermined antigen.Typically, an antigen binding molecule specifically binds to itspredetermined target with an affinity corresponding to a K_(D) of about10⁻⁷ M or less, and binds to the predetermined target with an affinitycorresponding to K_(D) that is at least 10 fold less, at least 100 foldless or at least 1000 fold less than its K_(D) for a non-specific andunrelated target (e.g., BSA, casein). In some embodiments, an antigenbinding molecule specifically binds to its predetermined target with anaffinity corresponding to a K_(D) of about 10⁻⁸ M or less, 10⁻⁹M or lessor 10⁻¹⁰ M or less.

As used herein, a “T cell receptor” or “TCR” refers to an antigenbinding protein in that includes both a TCR antigen binding domain(e.g., a TCR variable domain) and at least a portion of a TCR constantdomain. As used herein, a “constant domain” of a TCR polypeptideincludes a membrane-proximal TCR constant domain, and may also include aTCR transmembrane domain and/or a TCR cytoplasmic tail. In certainembodiments, the TCR is a soluble TCR and does not include a TCRtransmembrane domain or a TCR cytoplasmic tail. For example, in someembodiments, the TCR is a dimer that includes a first polypeptidecomprising a TCRβ variable domain linked to a TCRβ constant domain (or afragment thereof) and a second polypeptide comprising a TCRα linked to aTCRα constant domain (or a fragment thereof).

The term “unrearranged” includes the state of an immunoglobulin, TCR orCAR variable region locus or variable region gene segments wherein Vgene segments and J gene segments (for heavy or TCR(3 variable regions,D gene segments as well) are maintained separately but are capable ofbeing joined to form a rearranged V(D)J gene (a “variable region gene”)that comprises a single V, (D), J of the V(D)J repertoire.

Genetically Modified Non-Human Animals and ES Cells

In certain aspects, provided herein are non-human animals and ES cellscomprising in their genome an exogenous nucleic acid encoding TdT (e.g.,human, mouse or rat TdT). In certain embodiments, the genome of thenon-human animals and ES cells comprise further modifications including,for example, modifications that result in the expression of antigenbinding molecules having human variable domains (e.g., antibodies, TCRsand/or CARs).

The genetically modified non-human animals and ES cells provided hereincan be generated using any appropriate method known in the art. Forexample, non-human animal ES cells containing targeted geneticmodifications can be generated using VELOCIGENE® technology, which isdescribed in U.S. Pat. Nos. 6,586,251, 6,596,541, 7,105,348, andValenzuela et al. (2003) “High-throughput engineering of the mousegenome coupled with high-resolution expression analysis” Nat. Biotech.21(6): 652-659, and U.S Pat. Pub. No. US 2014/0310828, each of which ishereby incorporated by reference. Targeted modifications can also bemade using a CRISPR/Cas system, as described, for example, in U.S. Pat.No. 9,228,208, and U.S. Pub. Nos. US 2015-0159174 A1, US 2016-0060657A1, US 2015-0376650 A1, US 2015-0376651 A1, US 2016-0046960 A1, US2015-0376628 A1, and US 2016-0115486 A1, each of which is incorporatedby reference. Targeted modifications can also be made using ameganuclease, as described, for example, in U.S. Pat. Nos. 8,703,485,8,530,214 and 8,624,000, each of which is hereby incorporated byreference in its entirety. Non-targeted genetic modifications can bemade using standard methods in the art, including, for example, asdescribed in U.S. Pat. Nos. 6,150,584, 6,114,598, 5,633,425, 7,501,552,6,235,883, 6,998,514 and 5,776,773, each of which are herebyincorporated by reference in its entirety.

ES cells described herein can then be used to generate a non-humananimal using methods known in the art. For example, the mouse non-humananimal ES cells described herein can be used to generate geneticallymodified mice using the VELOCIMOUSE® method, as described in U.S. Pat.No. 7,294,754 and Poueymirou et al., Nature Biotech 25:91-99 (2007),each of which is hereby incorporated by reference. Rat ES cells can beused to generate modified rats using the method described, e.g., U.S.Pat. Pub. No. US 2014/0310828, incorporated herein by reference.Resulting mice or rats can be bread to homozygosity. Multiple distinctmodifications can be combined in a single genetically modified organismeither by breeding of separately modified animals or by introducingadditional modifications into an already modified ES cell (e.g., usingthe methods described herein).

In some embodiments, the non-human animal can be any non-human animal.In some embodiments, the non-human animal is a vertebrate. In someembodiments, the non-human animal is a mammal. In some embodiments, thegenetically modified non-human animal described herein may be selectedfrom a group consisting of a mouse, rat, rabbit, pig, bovine (e.g., cow,bull, buffalo), deer, sheep, goat, llama, chicken, cat, dog, ferret,primate (e.g., marmoset, rhesus monkey). For non-human animals wheresuitable genetically modifiable ES cells are not readily available,other methods can be employed to make a non-human animal comprising thegenetic modifications described herein. Such methods include, forexample, modifying a non-ES cell genome (e.g., a fibroblast or aninduced pluripotent cell) and employing nuclear transfer to transfer themodified genome to a suitable cell, such as an oocyte, and gestating themodified cell (e.g., the modified oocyte) in a non-human animal undersuitable conditions to form an embryo.

In some embodiments, the non-human animal is a mammal. In someembodiments, the non-human animal is a small mammal, e.g., of thesuperfamily Dipodoidea or Muroidea. In some embodiments, the non-humananimal is a rodent. In certain embodiments, the rodent is a mouse, a rator a hamster. In some embodiments, the rodent is selected from thesuperfamily Muroidea. In some embodiments, the non-human animal is froma family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(e.g., true mice and rats, gerbils, spiny mice, crested rats),Nesomyidae (e.g., climbing mice, rock mice, white-tailed rats, Malagasyrats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae(e.g., mole rates, bamboo rats, and zokors). In some embodiments, therodent is selected from a true mouse or rat (family Muridae), a gerbil,a spiny mouse, and a crested rat. In some embodiments, the mouse is froma member of the family Muridae. In some embodiments, the non-humananimal is a rodent. In some embodiments, the rodent is selected from amouse and a rat. In some embodiments, the non-human animal is a mouse.

In some embodiments, the non-human animal is a mouse of a C57BL strain.In some embodiments, the C57BL strain is selected from C57BL/A,C57BL/An, C57BL/GrFλ C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ,C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In someembodiments, the non-human animal is a mouse of a 129 strain. In someembodiments, the 129 strain is selected from the group consisting of astrain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV,129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac),129S7, 129S8, 129T1, 129T2. In some embodiments, the geneticallymodified mouse is a mix of a 129 strain and a C57BL strain. In someembodiments, the mouse is a mix of 129 strains and/or a mix of C57BL/6strains. In some embodiments, the 129 strain of the mix is a 129S6(129/SvEvTac) strain. In some embodiments, the mouse is a BALB strain(e.g., BALB/c). In some embodiments, the mouse is a mix of a BALB strainand another strain (e.g., a C57BL strain and/or a 129 strain). In someembodiments, the non-human animals provided herein can be a mousederived from any combination of the aforementioned strains.

In some embodiments, the non-human animal provided herein is a rat. Insome embodiments, the rat is selected from a Wistar rat, an LEA strain,a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. Insome embodiments, the rat strain is a mix of two or more strainsselected from the group consisting of Wistar, LEA, Sprague Dawley,Fischer, F344, F6, and Dark Agouti.

Non-human Animals Expressing Exogenous TdT

In certain aspects, provided herein are genetically modified non-humananimals and ES cells comprising in their germline and/or genome anucleic acid sequence encoding an exogenous TerminalDeoxynucleotidyltransferase (TdT). Deoxynucleotidyltransferase (TdT) isa DNA polymerase that catalyzes template-independent addition ofnucleotides (NP-additions) during junction formation in V(D)Jrecombination, which leads to an increase in antigen-receptor diversityin B and T lymphocytes. Template-independent additions, non-templateadditions, and non-germline additions all refer to nucleotide additionscatalyzed by TdT, and these terms are used herein interchangeably.

In certain embodiments, the sequence of the exogenous TdT in the genomeof the genetically modified non-human animal can be from any animal thatencodes a TdT or a TdT orthologue. In some embodiments, the TdT is avertebrate TdT. In some embodiments, the TdT is a mammalian TdT. In someembodiments, the TdT is from a mammal selected from a group consistingof a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer,sheep, goat, llama, chicken, cat, dog, ferret, primate (e.g., marmoset,rhesus monkey) or human. In some embodiments, the TdT is of endogenousspecies origin (i.e., the TdT sequence is that of the same species asthe genetically modified non-human animal). In some embodiments, the TdTis human TdT, mouse TdT or rat TdT. In some embodiments, the nucleicacid sequence is the genomic TdT sequence (i.e., including exons andintrons). In some embodiments, the nucleic acid sequence encodes TdTmRNA/cDNA (i.e., the exons of one or more TdT isoforms).

Human TdT (hTdT) is encoded by the DNTT gene, which is located on humanchromosome 10. An exemplary genomic DNA sequences of hTdT can be foundat position 96304328 to 96338564 of NCBI accession number NC_000010.11,which is hereby incorporated by reference. Exemplary mRNA sequence ofisoforms of hTdT is provided by NCBI accession numbers NM_001017520.1and NM_004088.3, each of which is hereby incorporated by reference. Theprotein sequences encoded by these isoforms is provided by NCBIaccession numbers NP_001017520.1 and NP_004079.3, respectively, each ofwhich is hereby incorporated by reference. Among the TdT isoforms is ashort isoform (hTdTS) and two long isoforms (hTdTL1 and hTdTL2). Thesequences of the three isoforms are provided, for example, in Thai andKearney, Adv. Immunol. 86:113-36 (2005), which is hereby incorporated byreference. In certain embodiments the exogenous nucleic acid sequenceencodes hTdTS. In some embodiments, the exogenous nucleic acid sequenceencodes hTdTL1. In some embodiments, the exogenous nucleic acid sequenceencodes hTdTL2. In certain embodiments, the non-human organism comprisesexogenous nucleic acid sequences encoding multiple isoforms (e.g., bothhTdTS and hTdTL2). In certain embodiments, the non-human organismcomprises exogenous nucleic acid sequences encoding all three humanisoforms (e.g., both hTdTS and hTdTL2).

Mouse TdT (mTdT) is encoded by the Dntt gene, which is located on mousechromosome 19. An exemplary genomic DNA sequence of mTdT can be found atposition 41029275 to 41059525 of NCBI accession number NC_000085.6,which is hereby incorporated by reference. Exemplary mRNA sequences ofisoforms of mTdT is provided by NCBI accession numbers NM_001043228.1and NM_009345.2, each of which is hereby incorporated by reference. Theprotein sequences encoded by these isoforms is provided by NCBIaccession numbers NP_001036693.1 and NP_033371.2, respectively, each ofwhich is hereby incorporated by reference.

Rat TdT (rTdT) is encoded by the Dntt gene, which is located on ratchromosome 1. An exemplary genomic DNA sequence of rTdT can be found atposition 260289626 to 260321174 of NCBI accession number NC_005100.4,which is hereby incorporated by reference. An exemplary mRNA sequence ofrTdT is provided by NCBI accession number NM_001012461.1, which ishereby incorporated by reference. The protein sequence encoded by thismRNA is provided by NCBI accession number NP_001012479.1, which ishereby incorporated by reference.

In some embodiments, the genome of the genetically modified non-humananimal contains multiple copies of the nucleic acid sequence encodingthe exogenous TdT. In some embodiments, the genetically modifiednon-human animal contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19 or 20 copies of the nucleic acid sequence encodingthe exogenous TdT. In some embodiments, the genetically modifiednon-human animal contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 copies of the nucleic acid sequenceencoding the exogenous TdT.

In some embodiments, the nucleic acid sequence encoding the exogenousTdT is operably linked to one or more transcriptional control element(e.g., a promoter and/or an enhancer). In some embodiments, thetranscriptional control element is a constitutive (i.e., ubiquitous)promoter. Examples of constitutive promoters include, but are notlimited to, a SV40, a CMV promoter, an adenoviral promoter, an EF1promoter, a β-actin promoter, an EGR1 promoter, an eIF4A1 promoter, aFerH promoter, a FerL promoter, a GAPD_(H) promoter, a GRP78 promoter, aGRP94 promoter, a HSP70 promoter, a β-Kin promoter, a PGK-1 promoter, aROSA promoter and an Ubiquitin B promoter. In some embodiments, thenucleic acid sequence is not operably linked to a constitutive promoter.

In some embodiments, the transcriptional control element inducesexpression of the encoded TdT during B cell development. In someembodiments, the transcriptional control element induces expression ofTdT in pro-B cells and/or pre-B cells. In some embodiments, thetranscriptional control element is a transcriptional control element(e.g., a promoter and/or enhancer) of a gene expressed during B celldevelopment, in pro-B cells and/or in pre-B cells. In some embodiments,the transcriptional control element is a RAG1 transcriptional controlelement, a RAG2 transcriptional control element, an immunoglobulin heavychain transcriptional control element, an immunoglobulin κ light chaintranscriptional control element and/or an immunoglobulin λ light chaintranscriptional control element. In some embodiment, the transcriptionalcontrol element is of endogenous species origin. In some embodiments,the transcriptional control element is a mouse transcriptional controlelement, a rat transcriptional control element or a humantranscriptional control element. In some embodiments, thetranscriptional control element is an endogenous transcriptional controlelement (e.g., the nucleotide sequence encoding the exogenous TdT isinserted into the non-human animal's genome at a position such thatexpression of the exogenous TdT is at least partially controlled by anendogenous transcriptional control element). In some embodiments,transcriptional control elements may include those regulatingtranscription of: RAG1, RAG2, λ5, VpreB, CD34, CD45, AA4.1, CD45R,IL-7R, MHC class II, CD10, CD19, CD38, CD20, CD40, variousimmunoglobulin light and heavy chain V gene segments promoters andenhancers (see. e.g., a list of various V gene segments listed on theInternational Immunogenetics Information System® website—IMGT, imgt.org,e.g., mouse V_(H)1-72 promoter and others, etc.). Transcriptionalcontrol elements may include those of human, mouse, rat, or otherspecies origin.

In some embodiments, the transcriptional control element inducesexpression of the encoded TdT during T cell development. In someembodiments, the transcriptional control element induces expression ofTdT in CD4/CD8 double-negative (DN) thymocytes and/or CD4/CD8double-positive (DP) thymocytes. In some embodiments, thetranscriptional control element is a transcriptional control element(e.g., a promoter and/or enhancer) of a gene expressed during T celldevelopment, in DN thymocytes and/or in DP thymocytes. In someembodiments, the transcriptional control element is a RAG1transcriptional control element, a RAG2 transcriptional control element,a TCRα transcriptional control element, a TCRβ transcriptional controlelement, a TCRγ transcriptional control element and/or a TCRδtranscriptional control element. In some embodiment, the transcriptionalcontrol element is of endogenous species origin. In some embodiments,the transcriptional control element is a mouse transcriptional controlelement, a rat transcriptional control element or a humantranscriptional control element. In some embodiments, thetranscriptional control element is an endogenous transcriptional controlelement (e.g., the nucleotide sequence encoding the exogenous TdT isinserted into the non-human animal's genome at a position such thatexpression of the exogenous TdT is at least partially controlled by anendogenous transcriptional control element). In some embodiments,transcriptional control elements may include those regulatingtranscription of: RAG1, RAG2, Lck, ZAP-70, CD34, CD2, HSA, CD44, CD25,PTλ CD4, CD8, CD69, various TCRα, TCRβ, TCRδ, and TCRγ V gene segmentspromoters and enhancers (see. e.g., a list of various V gene segmentslisted on the International Immunogenetics Information System®website—IMGT, imgt.org, etc.) Transcriptional control elements mayinclude those of human, mouse, rat, or other species origin.

In some embodiments, the nucleic acid encoding the TdT is located in thegenome of the non-human animal at or proximal to (e.g., within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 kb) agenomic locus of a gene that is expressed during B cell development, inpro-B cells and/or in pre-B cells. In some embodiments, the nucleic acidsequence encoding TdT is located at or proximal to an immunoglobulin κlight chain locus, an immunoglobulin λ light chain locus, animmunoglobulin heavy chain locus, a RAG1 locus or a RAG2 locus.

In some embodiments, the nucleic acid encoding the TdT is located in thegenome of the non-human animal at or proximal to (e.g., within 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 kb) agenomic locus of a gene that is expressed during T cell development, inDN thymocytes and/or in DP thymocytes. In some embodiments, the nucleicacid sequence encoding TdT is located at or proximal to a TCRα chainlocus, a TCRβ chain locus, a TCRγ chain locus, a TCRδ chain locus, aRAG1 locus or a RAG2 locus.

In some embodiments, the non-human animal provided herein expresseselevated levels of TdT expression during one or more stages to T celland/or B cell development (e.g., in pro-B cells, in pre-B cells, in DNthymocytes and/or in DP thymocytes) compared to a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the genetically modifiednon-human animals provided herein express at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100% 150%, 200%, 250%, 300%, 350%, 400%, 450%or 500% more TdT in one or more stages of T cell and/or B celldevelopment than a corresponding non-human animal.

In some embodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a lower percentageof V-J immunoglobulin κ chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctions notcontaining non-template additions in the genetically modified non-humananimals provided herein is less than percentage of V-J immunoglobulin κchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 1N-addition than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 1 N-addition in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 2 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 3N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 3 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 4N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 4 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-Jimmunoglobulin κ chain junctions in the animal comprise non-templateadditions. In some embodiments, the non-human animal has a greaterfrequency of unique immunoglobulin κ chain CDR3 sequences then acorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%,90% or 100%. In some embodiments, the non-human animal provided hereinhas at least 900, 1000, 1100, 1200, 1300, 1400, 1500 or 1700 uniqueimmunoglobulin κ chain CDR3 sequences per 10,000 immunoglobulin κ chainCDR3 sequences.

In some embodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a lower percentageof V-J immunoglobulin λ chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctions notcontaining non-template additions in the genetically modified non-humananimals provided herein is less than percentage of V-J immunoglobulin λchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 1N-addition than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 1 N-addition in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 2 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 3N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 3 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 4N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 4 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-Jimmunoglobulin λ chain junctions in the animal comprise non-templateadditions. In some embodiments, the non-human animal has a greaterfrequency of unique immunoglobulin λ chain CDR3 sequences then acorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%,90% or 100%. In some embodiments, the non-human animal provided hereinhas at least 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290 or 300 unique immunoglobulin λ chain CDR3 sequences per10,000 immunoglobulin λ chain CDR3 sequences.

In some embodiments, the non-human animal provided herein has a greaterpercentage of V-D immunoglobulin heavy chain junctions containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D immunoglobulin heavy chain junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-D immunoglobulinheavy chain junctions in the corresponding non-human animal by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a lower percentageof V-D immunoglobulin heavy chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D immunoglobulin heavy chain junctionsnot containing non-template additions in the genetically modifiednon-human animals provided herein is less than percentage of V-Dimmunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, the non-human animal provided hereinhas a greater percentage of V-D immunoglobulin heavy chain junctionscontaining at least 1 N-addition than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of V-D immunoglobulin heavychain junctions containing at least 1 N-addition in the geneticallymodified non-human animals provided herein is greater than percentage ofV-D immunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, the non-human animal provided hereinhas a greater percentage of V-D immunoglobulin heavy chain junctionscontaining at least 2 N-additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of V-D immunoglobulin heavychain junctions containing at least 2 N-additions in the geneticallymodified non-human animals provided herein is greater than percentage ofV-D immunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, the non-human animal provided hereinhas a greater percentage of V-D immunoglobulin heavy chain junctionscontaining at least 3 N-additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of V-D immunoglobulin heavychain junctions containing at least 3 N-additions in the geneticallymodified non-human animals provided herein is greater than percentage ofV-D immunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, the non-human animal provided hereinhas a greater percentage of V-D immunoglobulin heavy chain junctionscontaining at least 4 N-additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of V-D immunoglobulin heavychain junctions containing at least 4 N-additions in the geneticallymodified non-human animals provided herein is greater than percentage ofV-D immunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%or 40% of the V-D immunoglobulin heavy chain junctions in the animalcomprise non-template additions. In some embodiments, the non-humananimal has a greater frequency of unique immunoglobulin heavy chain CDR3sequences then a corresponding non-human animal by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%,50%, 60%, 70%, 80%, 90% or 100%.

In some embodiments, the non-human animal provided herein has a greaterpercentage of D-J immunoglobulin heavy chain junctions containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of D-J immunoglobulin heavy chain junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of D-J immunoglobulinheavy chain junctions in the corresponding non-human animal by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a lower percentageof D-J immunoglobulin heavy chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of D-J immunoglobulin heavy chain junctionsnot containing non-template additions in the genetically modifiednon-human animals provided herein is less than percentage of D-Jimmunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, the non-human animal provided hereinhas a greater percentage of D-J immunoglobulin heavy chain junctionscontaining at least 1 N-addition than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of D-J immunoglobulin heavychain junctions containing at least 1 N-addition in the geneticallymodified non-human animals provided herein is greater than percentage ofD-J immunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, the non-human animal provided hereinhas a greater percentage of D-J immunoglobulin heavy chain junctionscontaining at least 2 N-additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of D-J immunoglobulin heavychain junctions containing at least 2 N-additions in the geneticallymodified non-human animals provided herein is greater than percentage ofD-J immunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, the non-human animal provided hereinhas a greater percentage of D-J immunoglobulin heavy chain junctionscontaining at least 3 N-additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of D-J immunoglobulin heavychain junctions containing at least 3 N-additions in the geneticallymodified non-human animals provided herein is greater than percentage ofD-J immunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, the non-human animal provided hereinhas a greater percentage of D-J immunoglobulin heavy chain junctionscontaining at least 4 N-additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of D-J immunoglobulin heavychain junctions containing at least 4 N-additions in the geneticallymodified non-human animals provided herein is greater than percentage ofD-J immunoglobulin heavy chain junctions in the corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,30% or 40%. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%or 40% of the D-J immunoglobulin heavy chain junctions in the animalcomprise non-template additions.

In some embodiments, the non-human animal provided herein has a greaterpercentage of V-J TCRα chain junctions containing non-template additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-J TCRα chain junctions containing non-template additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-J TCRα chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a lower percentage of V-J TCRα chain junctions not containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J TCRα chain junctions not containingnon-template additions in the genetically modified non-human animalsprovided herein is less than percentage of V-J TCRα chain junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-J TCRαchain junctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-J TCRα chainjunctions containing at least 1 N-addition in the genetically modifiednon-human animals provided herein is greater than percentage of V-J TCRαchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J TCRα chain junctions containing at least 2 N-additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-J TCRα chain junctions containing at least 2 N-additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-J TCRα chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of V-J TCRα chain junctions containingat least 3 N-additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J TCRα chain junctions containing atleast 3 N-additions in the genetically modified non-human animalsprovided herein is greater than percentage of V-J TCRα chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-J TCRαchain junctions containing at least 4 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-J TCRα chainjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-J TCRαchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-JTCRα chain junctions in the animal comprise non-template additions. Insome embodiments, the non-human animal has a greater frequency of uniqueTCRα CDR3 sequences then a corresponding non-human animal by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%.

In some embodiments, the non-human animal provided herein has a greaterpercentage of V-D TCRβ chain junctions containing non-template additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D TCRβ chain junctions containing non-template additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-D TCRβ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a lower percentage of V-D TCRβ chain junctions not containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D TCRβ chain junctions not containingnon-template additions in the genetically modified non-human animalsprovided herein is less than percentage of V-D TCRβ chain junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D TCRβchain junctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D TCRβ chainjunctions containing at least 1 N-addition in the genetically modifiednon-human animals provided herein is greater than percentage of V-D TCRβchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-D TCRβ chain junctions containing at least 2 N-additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D TCRβ chain junctions containing at least 2 N-additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-D TCRβ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of V-D TCRβ chain junctions containingat least 3 N-additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D TCRβ chain junctions containing atleast 3 N-additions in the genetically modified non-human animalsprovided herein is greater than percentage of V-D TCRβ chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D TCRβchain junctions containing at least 4 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D TCRβ chainjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-D TCRβchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-DTCRβ chain junctions in the animal comprise non-template additions. Insome embodiments, the non-human animal has a greater frequency of uniqueTCRβ CDR3 sequences then a corresponding non-human animal by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%.

In some embodiments, the non-human animal provided herein has a greaterpercentage of D-J TCRβ chain junctions containing non-template additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of D-J TCRβ chain junctions containing non-template additionsin the genetically modified non-human animals provided herein is greaterthan percentage of D-J TCRβ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a lower percentage of D-J TCRβ chain junctions not containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of D-J TCRβ chain junctions not containingnon-template additions in the genetically modified non-human animalsprovided herein is less than percentage of D-J TCRβ chain junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of D-J TCRβchain junctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of D-J TCRβ chainjunctions containing at least 1 N-addition in the genetically modifiednon-human animals provided herein is greater than percentage of D-J TCRβchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of D-J TCRβ chain junctions containing at least 2 N-additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of D-J TCRβ chain junctions containing at least 2 N-additionsin the genetically modified non-human animals provided herein is greaterthan percentage of D-J TCRβ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of D-J TCRβ chain junctions containingat least 3 N-additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of D-J TCRβ chain junctions containing atleast 3 N-additions in the genetically modified non-human animalsprovided herein is greater than percentage of D-J TCRβ chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of D-J TCRβchain junctions containing at least 4 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of D-J TCRβ chainjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of D-J TCRβchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the D-JTCRβ chain junctions in the animal comprise non-template additions.

In some embodiments, the non-human animal provided herein has a greaterpercentage of V-J TCRγ chain junctions containing non-template additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-J TCRγ chain junctions containing non-template additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-J TCRγ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a lower percentage of V-J TCRγ chain junctions not containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J TCRγ chain junctions not containingnon-template additions in the genetically modified non-human animalsprovided herein is less than percentage of V-J TCRγ chain junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-J TCRγchain junctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-J TCRγ chainjunctions containing at least 1 N-addition in the genetically modifiednon-human animals provided herein is greater than percentage of V-J TCRγchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J TCRγ chain junctions containing at least 2 N-additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-J TCRγ chain junctions containing at least 2 N-additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-J TCRγ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of V-J TCRγ chain junctions containingat least 3 N-additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J TCRγ chain junctions containing atleast 3 N-additions in the genetically modified non-human animalsprovided herein is greater than percentage of V-J TCRγ chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-J TCRγchain junctions containing at least 4 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-J TCRγ chainjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-J TCRγchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-JTCRγ chain junctions in the animal comprise non-template additions. Insome embodiments, the non-human animal has a greater frequency of uniqueTCRγ CDR3 sequences then a corresponding non-human animal by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%.

In some embodiments, the non-human animal provided herein has a greaterpercentage of V-D TCRδ chain junctions containing non-template additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D TCRδ chain junctions containing non-template additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-D TCRδ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a lower percentage of V-D TCRδ chain junctions not containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D TCRδ chain junctions not containingnon-template additions in the genetically modified non-human animalsprovided herein is less than percentage of V-D TCRδ chain junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D TCRδchain junctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D TCRδ chainjunctions containing at least 1 N-addition in the genetically modifiednon-human animals provided herein is greater than percentage of V-D TCRδchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-D TCRδ chain junctions containing at least 2 N-additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D TCRδ chain junctions containing at least 2 N-additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-D TCRδ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of V-D TCRδ chain junctions containingat least 3 N-additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D TCRδ chain junctions containing atleast 3 N-additions in the genetically modified non-human animalsprovided herein is greater than percentage of V-D TCRδ chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D TCRδchain junctions containing at least 4 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D TCRδ chainjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-D TCRδchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-DTCRδ chain junctions in the animal comprise non-template additions. Insome embodiments, the non-human animal has a greater frequency of uniqueTCRδ CDR3 sequences then a corresponding non-human animal by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%.

In some embodiments, the non-human animal provided herein has a greaterpercentage of D-J TCRδ chain junctions containing non-template additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of D-J TCRδ chain junctions containing non-template additionsin the genetically modified non-human animals provided herein is greaterthan percentage of D-J TCRδ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a lower percentage of D-J TCRδ chain junctions not containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of D-J TCRδ chain junctions not containingnon-template additions in the genetically modified non-human animalsprovided herein is less than percentage of D-J TCRδ chain junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of D-J TCRδchain junctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of D-J TCRδ chainjunctions containing at least 1 N-addition in the genetically modifiednon-human animals provided herein is greater than percentage of D-J TCRδchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of D-J TCRδ chain junctions containing at least 2 N-additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of D-J TCRδ chain junctions containing at least 2 N-additionsin the genetically modified non-human animals provided herein is greaterthan percentage of D-J TCRδ chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of D-J TCRδ chain junctions containingat least 3 N-additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of D-J TCRδ chain junctions containing atleast 3 N-additions in the genetically modified non-human animalsprovided herein is greater than percentage of D-J TCRδ chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of D-J TCRδchain junctions containing at least 4 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of D-J TCRδ chainjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of D-J TCRδchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the D-JTCRδ chain junctions in the animal comprise non-template additions.

In some embodiments, the endogenous TdT loci in the non-human organismis intact. In some embodiments the endogenous TdT loci is inactivated.For example, in some embodiments, the endogenous TdT loci is deleted inwhole or in part such that the non-human organism does not expressendogenous TdT.

Non-Human Animals that Expressing Human Variable Domain Antibodies andExogenous TdT

In certain embodiments, the genetically modified non-human animals andnon-human animal ES cells comprising exogenous TdT as described hereinalso comprise in their germline and/or genome an immunoglobulin locus(exogenous or endogenous) containing an immunoglobulin variable regioncomprising unrearranged human immunoglobulin variable region genesegments and an immunoglobulin constant region comprising animmunoglobulin constant region gene and in which the unrearranged humanimmunoglobulin variable region gene segments are operably linked to theimmunoglobulin constant region gene. In some embodiments, the non-humananimals and non-human ES cells comprise in their germline and/or genomemultiple such immunoglobulin loci. For example, in some embodiments, thegenetically modified non-human animals and non-human animal ES cellscomprise in their germline and/or genome at least one immunoglobulinlocus comprising unrearranged human heavy chain variable region genesegments and at least one immunoglobulin locus comprising unrearrangedhuman light chain variable region gene segments (e.g., κ chain genesegments and/or λ chain gene segments). In some embodiments, thegenetically modified non-human animals and non-human animal ES cellscomprise in their germline and/or genome at least one immunoglobulinlocus comprising unrearranged human heavy chain variable region genesegments, at least one immunoglobulin locus comprising unrearrangedhuman κ chain variable region gene segments and at least oneimmunoglobulin locus comprising unrearranged human λ chain variableregion gene segments. In some embodiments, genetically modifiednon-human animals, e.g., genetically modified mice or rats, comprise intheir germline and/or genome genetically modified immunoglobulin loci(genetically modified rearranged or unrearranged immunoglobulin loci)such that the mice make human, humanized, partially human, reversechimeric (human variable and non-human constant regions) antibodies.

Immunoglobulin loci comprising human variable region gene segments areknown in the art and can be found, for example, in U.S. Pat. Nos.5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584,6,998,514, 7,795,494, 7,910,798, 8,232,449, 8,502,018, 8,697,940,8,703,485, 8,754,287, 8,791,323, 8,907,157, 9,035,128, 9,145,588, and9,206,263 and each of which is hereby incorporated by reference in itsentirety, as well as in U.S. Pat. Pub. Nos. 2008/0098490, 2010/0146647,2011/0195454, 2012/0167237, 2013/0145484, 2013/0167256, 2013/0219535,2013/0326647, 2013/0096287, 2014/013275, 2014/093908, and 2015/0113668,each of which is hereby incorporated by reference in its entirety, andin PCT Pub. Nos. WO2007117410, WO2008151081, WO2009157771, WO2010039900,WO2011004192, WO2011123708 and WO2014093908, each of which are herebyincorporated by reference in its entirety.

In some embodiments, the human unrearranged immunoglobulin variableregion gene segments are heavy chain gene segments and theimmunoglobulin constant region gene is a heavy chain constant regiongene. In some embodiments, the human unrearranged immunoglobulinvariable region gene segments are light chain, e.g., κ chain, genesegments, and the immunoglobulin constant region gene is a heavy chainconstant region gene.

In some embodiments, the human unrearranged immunoglobulin variableregion gene segments are heavy chain gene segments and theimmunoglobulin constant region gene is a κ chain constant region gene.In some embodiments, the human unrearranged immunoglobulin variableregion gene segments are κ chain gene segments and the immunoglobulinconstant region gene is a κ chain constant region gene. In someembodiments, the human unrearranged immunoglobulin variable region genesegments are λ chain gene segments and the immunoglobulin constantregion gene is a κ chain constant region gene. In some embodiments, thehuman unrearranged immunoglobulin variable region gene segments are λchain gene segments and the immunoglobulin constant region gene is λchain constant region gene.

In certain embodiments, the immunoglobulin variable region containsunrearranged human Ig heavy chain variable region gene segments. In someembodiments, the unrearranged human Ig variable region gene segmentscomprise a plurality of human V_(H) segments, one or more human D_(H)segments and one or more human J_(H) segments. In some embodiments, theunrearranged human Ig variable region gene segments comprise at least 3V_(H) gene segments, at least 18 V_(H) gene segments, at least 20 V_(H)gene segments, at least 30 V_(H) gene segments, at least 40 V_(H) genesegments, at least 50 V_(H) gene segments, at least 60 V_(H) genesegments, at least 70 V_(H) gene segments, or at least 80 V_(H) genesegments. In some embodiments, the unrearranged human Ig gene segmentsinclude all of the human D_(H) gene segments. In some embodiments, theunrearranged human Ig gene segments include all of the human JH genesegments. Exemplary variable regions comprising Ig heavy chain genesegments are provided, for example, in Macdonald et al., Proc. Natl.Acad. Sci. USA 111:5147-52 and supplemental information, which is herebyincorporated by reference. In some embodiments, the non-human animalsprovided herein have a restricted immunoglobulin heavy chain locuscharacterized by a single polymorphic human V_(H) gene segment, aplurality of D_(H) gene segments and a plurality of J_(H) gene segments(e.g., as described in U.S. Pat. Pub. No. 2013/0096287, which is herebyincorporated by reference). In some embodiments the V_(H) gene segmentis VH1-2 or VH1-69.

In various embodiments, the immunoglobulin locus modifications describedherein do not affect fertility of the non-human animal. In someembodiments, the heavy chain locus comprises an endogenous Adam6a gene,Adam6b gene, or both, and the genetic modification does not affect theexpression and/or function of the endogenous Adam6a gene, Adam6b gene,or both. In some embodiments, the genome of the genetically modifiednon-human animal comprises an ectopically located Adam6a gene, Adam6bgene, or both. Exemplary non-human animals expressing exogenous Adam6aand/or Adam6b are described in U.S. Pat. Nos. 8,642,835 and 8,697,940,each of which is hereby incorporated by reference in its entirety.

In some embodiments, the human immunoglobulin heavy chain variableregion gene segments rearrange during B cell development to generaterearranged human heavy chain variable region genes in the B cells of thenon-human organism. In some embodiments, the non-human animal providedherein has a greater percentage of V-D and/or D-J immunoglobulin heavychain junctions containing non-template additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-Jimmunoglobulin heavy chain junctions containing non-template additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-D and/or D-J immunoglobulin heavy chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a lower percentage of V-Dimmunoglobulin heavy chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D and/or D-J immunoglobulin heavy chainjunctions not containing non-template additions in the geneticallymodified non-human animals provided herein is less than percentage ofV-D and/or D-J immunoglobulin heavy chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of V-D and/or D-J immunoglobulin heavychain junctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-Jimmunoglobulin heavy chain junctions containing at least 1 N-addition inthe genetically modified non-human animals provided herein is greaterthan percentage of V-D and/or D-J immunoglobulin heavy chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D and/orD-J immunoglobulin heavy chain junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D and/or D-J immunoglobulin heavy chainjunctions containing at least 2 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-Dand/or D-J immunoglobulin heavy chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of V-D and/or D-J immunoglobulin heavychain junctions containing at least 3 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-Jimmunoglobulin heavy chain junctions containing at least 3 N-additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-D and/or D-J immunoglobulin heavy chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D and/orD-J immunoglobulin heavy chain junctions containing at least 4N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D and/or D-J immunoglobulin heavy chainjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-Dand/or D-J immunoglobulin heavy chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, at least 10%, 15%, 20%, 25%,30%, 35% or 40% of the V-D and/or D-J immunoglobulin heavy chainjunctions in the animal comprise non-template additions. In someembodiments, the non-human animal has a greater frequency of uniqueimmunoglobulin heavy chain CDR3 sequences then a corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%.

In certain embodiments, the immunoglobulin variable region containsunrearranged human Ig κ variable region gene segments. In someembodiments, the unrearranged human immunoglobulin variable region genesegments comprise a plurality of human V_(κ) segments and one or morehuman J_(κ) segments. In some embodiments, the unrearranged humanimmunoglobulin variable region gene segments comprise all of the humanJκ segments. In some embodiments, the immunoglobulin variable regiongene segments comprise four functional Vκ segments and all human J_(κ)segments. In some embodiments, the immunoglobulin variable region genesegments comprise 16 functional V_(κ) segments and all human J_(κ)segments (e.g., all functional human Vκ segments and J_(κ) segments). Insome embodiments, the unrearranged human immunoglobulin variable regiongene segments comprise all of the human Vκ segments and all human J_(κ)segments. Exemplary variable regions comprising Ig κ gene segments areprovided, for example, in Macdonald et al., Proc. Natl. Acad. Sci. USA111:5147-52 and supplemental information, which is hereby incorporatedby reference. In some embodiments, the non-human animals provided hereinhave a restricted immunoglobulin light chain locus characterized by nomore than two human V_(L) gene segments and a plurality of J_(L) genesegments (e.g., dual light chain mice, or DLC, as described in U.S. Pat.Pub. No. 2013/0198880, which is hereby incorporated by reference). Insome embodiments the V_(L) gene segments are V_(κ) gene segments. Insome embodiments the V_(L) gene segments are V_(λ) gene segments. Insome embodiments the V_(κ) gene segments are IGKV3-20 and IGKV1-39.

In some embodiments, the human immunoglobulin κ variable region genesegments rearrange during B cell development to generate rearrangedhuman κ variable region genes in the B cells of the non-human organism.In some embodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a lower percentageof V-J immunoglobulin κ chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctions notcontaining non-template additions in the genetically modified non-humananimals provided herein is less than percentage of V-J immunoglobulin κchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 1N-addition than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 1 N-addition in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin chain junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 2 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 3N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 3 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 4N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 4 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-Jimmunoglobulin κ chain junctions in the animal comprise non-templateadditions. In some embodiments, the non-human animal has a greaterfrequency of unique immunoglobulin κ chain CDR3 sequences then acorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%,90% or 100%. In some embodiments, the non-human animal provided hereinhas at least 900, 1000, 1100, 1200, 1300, 1400, 1500 or 1700 uniqueimmunoglobulin κ chain CDR3 sequences per 10,000 immunoglobulin κ chainCDR3 sequences.

In certain embodiments, the immunoglobulin variable region containsunrearranged human Ig λ variable region gene segments. In someembodiments, the unrearranged human immunoglobulin variable region genesegments comprise a plurality of human V_(λ) segments and one or morehuman J_(λ) segments. In some embodiments, the unrearranged humanimmunoglobulin variable region gene segments comprise all of the humanV_(λ) segments. In some embodiments, the unrearranged humanimmunoglobulin variable region gene segments comprise all of the humanh. segments. Exemplary variable regions comprising Ig λ gene segmentsare provided, for example, U.S. Pat. Pub. Nos. 2012/0073004 and2002/0088016, each of which is hereby incorporated by reference.

In some embodiments, the human immunoglobulin λ variable region genesegments rearrange during B cell development to generate rearrangedhuman λ variable region genes in the B cells of the non-human organism.In some embodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a lower percentageof V-J immunoglobulin λ chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctions notcontaining non-template additions in the genetically modified non-humananimals provided herein is less than percentage of V-J immunoglobulin λchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 1N-addition than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 1 N-addition in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 2 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 3N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 3 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 4N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 4 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-Jimmunoglobulin λ chain junctions in the animal comprise non-templateadditions. In some embodiments, the non-human animal has a greaterfrequency of unique immunoglobulin λ chain CDR3 sequences then acorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%,90% or 100%. In some embodiments, the non-human animal provided hereinhas at least 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290 or 300 unique immunoglobulin λ chain CDR3 sequences per10,000 immunoglobulin λ chain CDR3 sequences.

In some embodiments, the immunoglobulin variable region comprisingunrearranged human immunoglobulin variable region gene segments alsoincludes human immunoglobulin variable region intergenic sequences. Insome embodiments, the immunoglobulin variable region includes non-human(e.g., rodent, rat, mouse) Ig variable region intergenic sequences. Insome embodiments, the intergenic sequence is of endogenous speciesorigin.

In some embodiments, the non-human organism comprises in its germlineand/or genome an immunoglobulin locus comprising a rearranged heavychain variable region (a universal heavy chain variable region). In someembodiments, the rearranged Ig heavy chain variable region gene is ahuman rearranged Ig heavy chain variable region gene. Exemplaryrearranged Ig heavy chain variable regions are provided in U.S. PatentPub. No. 2014/0245468, which is hereby incorporated by reference. Insome embodiments, the non-human organism comprising a universal heavychain variable region is used to produce bispecific antibodies.

In some embodiments, the non-human organism comprises in its germlineand/or genome an immunoglobulin locus comprising a rearranged lightvariable region (a universal light chain variable region). In someembodiments, the rearranged Ig light chain variable region gene is ahuman rearranged Ig light chain variable region gene. Exemplaryrearranged Ig light chain variable regions are provided in, e.g., U.S.Patent Pub. Nos. 2011/0195454, 2012/0021409, 2012/0192300, 2013/0045492,2013/0185821, 2013/0302836, 2015/0313193, 2015/0059009, and2013/0198879, which are hereby incorporated by reference. In someembodiments, the non-human organism (“universal light chain” organism)comprising a universal light chain variable region is used to producebispecific antibodies.

In some embodiments, the non-human organism comprises in its germlineand/or genome a light chain immunoglobulin locus comprising a limitedrepertoire of light chain variable gene segments (e.g., a dual lightchain variable region comprising two light chain variable genesegments). In some embodiments, the light chain variable gene segmentsin the limited repertoire of light chain gene segments are a human lightchain gene segments. Exemplary dual light chain variable regions areprovided in U.S. Patent Pub. No. 2013/0198880, which is herebyincorporated by reference. In some embodiments, the non-human organismcomprising a dual light chain variable region is used to producebispecific antibodies.

In yet other embodiments, the non-human organism may comprise in itsgermline and/or genome a light chain and/or a heavy chain immunoglobulinlocus that includes insertions and/or replacements of histidine codonsdesigned to introduce pH-dependent binding properties to the antibodiesgenerated in such non-human organism. In some of such embodiments, thehistidine codons are inserted and/or replaced in the nucleic acidsequences encoding CDR3. Various such light and/or heavy immunoglobulinloci are provided in U.S. Pat. Nos. 9,301,510, 9,334,334, U.S. PatentApplication Publication Nos. 2013/0247236, 20140013456, incorporatedherein by reference.

In some embodiments, the immunoglobulin constant region comprises aheavy chain constant region gene. In some embodiments, the heavy chainconstant region gene is a human heavy chain constant region gene. Insome embodiments, the heavy chain constant region gene is of endogenousspecies origin. In some embodiments, the heavy chain constant regiongene is a mouse constant region gene or a rat constant region gene. Insome embodiments, the constant region gene is a mixture of human andnon-human sequence. For example, in some embodiments, the constantregion gene encodes a human CH1 region and a non-human (e.g., endogenousspecies origin, mouse, rat) CH2 and/or CH3 region. In some embodiments,the heavy chain constant region gene is an Cμ, Cδ, Cγ (Cγ1, Cγ2, Cγ3,Cγ4), Cα or Cϵ constant region gene. In some embodiments, the constantregion gene is an endogenous constant region gene. In some embodiments,the constant region gene encodes a mutated CH1 region so that thenon-human animal expresses heavy chain only antibodies (see., e.g., U.S.Pat. No. 8,754,287, U.S. Patent Application Publication No.2015/0289489, incorporated herein by reference). In some embodiments,e.g., where the goal is to generate heavy chains to make bispecificantibodies (e.g., in universal or dual light chain organisms), the Fcdomains of the heavy chains comprise modifications to facilitate heavychain heterodimer formation and/or to inhibit heavy chain homodimerformation. Such modifications are provided, for example, in U.S. Pat.Nos. 5,731,168, 5,807,706, 5,821,333, 7,642,228 and 8,679,785 and inU.S. Pat. Pub. No. 2013/0195849, each of which is hereby incorporated byreference.

In some embodiments, the immunoglobulin constant region comprises alight chain constant region gene. In some embodiments, the light chainconstant region gene is a κ constant region gene. In some embodiments,the light chain constant region gene is a λ constant region gene. Insome embodiments, the light chain constant region gene is of endogenousspecies origin. In some embodiments, the light chain constant regiongene is a mouse constant region gene or a rat constant region gene. Insome embodiments, the light chain constant region gene is a mixture ofhuman and non-human sequence.

In some embodiments, the immunoglobulin variable region comprising humanvariable region gene segments and the immunoglobulin constant regiongene to which the variable region gene segments are operably linked arelocated at an endogenous immunoglobulin locus. In some embodiments, theendogenous immunoglobulin locus is an endogenous heavy chain locus. Insome embodiments, the endogenous immunoglobulin locus is an endogenous κlocus. In some embodiments, the endogenous immunoglobulin locus is anendogenous λ locus. In some embodiments, the constant region gene towhich the human variable region gene segments are operably linked is anendogenous constant region gene.

In some embodiments, one or more of the endogenous immunoglobulin locior a portion of the one or more endogenous loci (e.g., a variable regionand/or a constant region) in the genome of the non-human animal providedherein is inactivated. Endogenous immunoglobulin variable region geneloci and portions thereof can be inactivated using any method known inthe art, including, but not limited to, the deletion of the locus or aportion thereof from the genome of the organism, the replacement of alocus or a portion thereof with a different nucleic acid sequence, theinversion of a portion of the locus and/or the displacement of a portionof the locus to another position in the genome of the non-humanorganism. In some embodiments the inactivation of the locus is only apartial inactivation. In some embodiments, the variable region of thelocus is inactivated but the constant region remains functional (e.g.,because it is operably linked to non-endogenous variable region genesegments).

In some embodiments, the genetically modified non-human animal includesan inactivated endogenous immunoglobulin heavy chain locus. In someembodiments, the endogenous immunoglobulin heavy chain locus or aportion thereof is inactivated by deletion, replacement, displacementand/or inversion of at least part of the endogenous variable region ofthe endogenous heavy chain locus. In some embodiments, the at least partof the variable region of the endogenous heavy chain locus that isdeleted, replaced, displaced, and/or inverted comprises the J segmentsof the variable region. In some embodiments, the endogenousimmunoglobulin heavy chain locus or portion thereof is inactivated bydeletion, replacement, displacement and/or inversion of at least part ofthe endogenous constant region of the endogenous heavy chain locus. Insome embodiments, the at least part of the constant region of theendogenous heavy chain locus that is deleted, replaced, displaced,and/or inverted comprises the Cμ gene of the endogenous constant region.

In some embodiments, the genetically modified non-human animal includesan inactivated endogenous immunoglobulin κ chain locus. In someembodiments, the endogenous immunoglobulin κ chain locus or a portionthereof is inactivated by deletion, replacement, displacement and/orinversion of at least part of the endogenous variable region of theendogenous κ chain locus. In some embodiments, the at least part of thevariable region of the endogenous κ chain locus that is deleted,replaced, displaced, and/or inverted comprises the J segments of thevariable region. In some embodiments, the endogenous immunoglobulin κchain locus or portion thereof is inactivated by deletion, replacement,displacement and/or inversion of at least part of the endogenousconstant region of the endogenous κ chain locus. In some embodiments,the at least part of the constant region of the endogenous κ chain locusthat is deleted, replaced, displaced, and/or inverted comprises theC_(κ) gene of the endogenous constant region.

In some embodiments, the genetically modified non-human animal includesan inactivated endogenous immunoglobulin λ chain locus. In someembodiments, the endogenous immunoglobulin λ chain locus or a portionthereof is inactivated by deletion, replacement, displacement and/orinversion of at least part of an endogenous variable region of theendogenous λ chain locus. In some embodiments, the at least part of atleast one V-J-C gene cluster in the endogenous λ chain locus is deleted,replaced, displaced, and/or inverted. In some embodiments, theendogenous immunoglobulin λ chain locus or portion thereof isinactivated by deletion, replacement, displacement and/or inversion ofat least part of an endogenous constant region of the endogenous λ chainlocus. In some embodiments, the at least part of the constant region ofthe endogenous λ chain locus that is deleted, replaced, displaced,and/or inverted comprises a Cλ gene of the endogenous constant region.

In some embodiments, the genetically modified non-human animal providedherein expresses antibodies having human variable domains (e.g., a humanvariable domain derived from the unrearranged human variable region genesegments described herein). In some embodiments, the human variabledomain is a human heavy chain variable domain. In some embodiments, theantibodies are heavy chain only antibodies. In some embodiments, thehuman variable domain is a human light chain variable domain. In someembodiments, the antibodies produced by the non-human animals have bothhuman heavy chain variable domains and human light chain variabledomains. In some embodiments, the antibodies have human heavy chainconstant domains. In some embodiments, the antibodies have human lightchain constant domains. In some embodiments, the heavy and/or lightchain constant domain is of non-human origin. For example, in someembodiments, the heavy chain constant domain is of endogenous speciesorigin. In some embodiments, the heavy chain constant domain is of mouseor rat origin. In some embodiments, the light chain constant domain isof endogenous species origin. In some embodiments, the light chainconstant domain is of rat or mouse origin.

Non-human Animals Expressing Human Variable Domain T Cell Receptors andExogenous TdT

In certain embodiments, the genetically modified non-human animals andnon-human animal ES cells that comprise exogenous TdT as describedherein also comprise in their germline and/or genome a TCRδ locus(exogenous or endogenous) containing a TCR variable region comprisingunrearranged human TCR variable region gene segments and a TCR constantregion comprising a TCR constant region gene and in which theunrearranged human TCR variable region gene segments are operably linkedto the TCR constant region gene. In some embodiments, variousgenetically modified non-human animals, e.g., genetically modified mice,comprise in their germline and/or genome genetically modified T cellreceptor loci (genetically modified TCRα, β, γ and/or δ loci) such thatthe mice express human, humanized, partially human, reverse chimeric(human variable and non-human constant regions) T cell receptors. In oneembodiment, the exemplary non-human animal is provided in U.S. Pat. No.9,113,616 and International Pub. No. WO 2016/164492, incorporated hereinby reference.

In some embodiments, the TCR constant region gene is a non-human TCRconstant region gene. In some embodiments, the TCR constant region geneis a rodent constant region gene, such as a rat constant region gene ora mouse constant region gene. In some embodiments, the constant regiongene is of endogenous species origin. In some embodiments, the TCRconstant region gene is a human constant region gene.

In some embodiments, the non-human animals and non-human ES cellscomprise in their germline and/or genome multiple such TCR loci. Forexample, in some embodiments, the genetically modified non-human animalsand non-human animal ES cells comprise in their germline and/or genomeat least one TCRδ locus comprising unrearranged TCRα variable regiongene segments and at least one TCRδ locus comprising unrearranged TCRβvariable region gene segments. In some embodiments, the geneticallymodified non-human animals and non-human animal ES cells comprise intheir germline and/or genome at least one TCRδ locus comprisingunrearranged human TCRγ variable region gene segments and at least oneTCRδ locus comprising unrearranged human TCRδ variable region genesegments.

In some embodiments, the human unrearranged TCR variable region genesegments are TCRα gene segments and the TCR constant region gene is aTCRα constant region gene. In some embodiments, the human unrearrangedTCR variable region gene segments are TCRβ chain gene segments and theTCR constant region gene is a TCRβ constant region gene. In someembodiments, the human unrearranged TCR variable region gene segmentsare TCRγ chain gene segments and the TCR constant region gene is a TCRγconstant region gene. In some embodiments, the human unrearranged TCRvariable region gene segments are TCRδ chain gene segments and the TCRconstant region gene is a TCRδ constant region gene. Exemplary variableregions comprising human TCR gene segments are provided, for example, inU.S. Pat. No. 9,113,616 and Li et al., Nature Medicine 16:1029-1035(2010), each of which is hereby incorporated by reference.

In some embodiments, the TCR variable region contains unrearranged humanTCRβ variable region gene segments. In some embodiments, the human TCRβvariable region gene segments rearrange during T cell development togenerate rearranged human TCRβ variable region genes in the T cells ofthe non-human organism. In some embodiments, the non-human animalprovided herein has a greater percentage of V-D and/or D-J TCRβjunctions containing non-template additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-JTCRβ junctions containing non-template additions in the geneticallymodified non-human animals provided herein is greater than percentage ofV-D and/or D-J TCRβ junctions in the corresponding non-human animal byat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%.In some embodiments, the non-human animal provided herein has a lowerpercentage of V-D TCRβ junctions not containing non-template additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D and/or D-J TCRβ junctions not containing non-templateadditions in the genetically modified non-human animals provided hereinis less than percentage of V-D and/or D-J TCRβ junctions in thecorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, the non-humananimal provided herein has a greater percentage of V-D and/or D-J TCRβjunctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-JTCRβ junctions containing at least 1 N-addition in the geneticallymodified non-human animals provided herein is greater than percentage ofV-D and/or D-J TCRβ junctions in the corresponding non-human animal byat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%.In some embodiments, the non-human animal provided herein has a greaterpercentage of V-D and/or D-J TCRβ junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D and/or D-J TCRβ junctions containingat least 2 N-additions in the genetically modified non-human animalsprovided herein is greater than percentage of V-D and/or D-J TCRβjunctions in the corresponding non-human animal by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments,the non-human animal provided herein has a greater percentage of V-Dand/or D-J TCRβ junctions containing at least 3 N-additions than acorresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D and/or D-J TCRβ junctions containing at least 3N-additions in the genetically modified non-human animals providedherein is greater than percentage of V-D and/or D-J TCRβ junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D and/orD-J TCRβ junctions containing at least 4 N-additions than acorresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D and/or D-J TCRβ junctions containing at least 4N-additions in the genetically modified non-human animals providedherein is greater than percentage of V-D and/or D-J TCRβ junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, at least10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-D and/or D-J TCRβ junctionsin the animal comprise non-template additions. In some embodiments, thenon-human animal has a greater frequency of unique TCRβ CDR3 sequencesthen a corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%,70%, 80%, 90% or 100%.

In some embodiments, the TCR variable region contains unrearranged humanTCRα variable region gene segments. In some embodiments, the human TCRαvariable region gene segments rearrange during T cell development togenerate rearranged human TCRα variable region genes in the T cells ofthe non-human organism. In some embodiments, the non-human animalprovided herein has a greater percentage of V-J TCRα junctionscontaining non-template additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of V-J TCRα junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J TCRα junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a lower percentage of V-D TCRαjunctions not containing non-template additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-J TCRαjunctions not containing non-template additions in the geneticallymodified non-human animals provided herein is less than percentage ofV-J TCRα junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J TCRα junctions containing at least 1 N-addition than acorresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-J TCRα junctions containing at least 1 N-addition in thegenetically modified non-human animals provided herein is greater thanpercentage of V-J TCRα junctions in the corresponding non-human animalby at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or40%. In some embodiments, the non-human animal provided herein has agreater percentage of V-J TCRα junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J TCRα junctions containing at least 2N-additions in the genetically modified non-human animals providedherein is greater than percentage of V-J TCRα junctions in thecorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, the non-humananimal provided herein has a greater percentage of V-J TCRα junctionscontaining at least 3 N-additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of V-J TCRα junctionscontaining at least 3 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J TCRα junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-J TCRαjunctions containing at least 4 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-J TCRαjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-J TCRαjunctions in the corresponding non-human animal by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments,at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-J TCRα junctionsin the animal comprise non-template additions. In some embodiments, thenon-human animal has a greater frequency of unique TCRα CDR3 sequencesthen a corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%,70%, 80%, 90% or 100%.

In some embodiments, the TCR variable region contains unrearranged humanTCRδ variable region gene segments. In some embodiments, the human TCRδvariable region gene segments rearrange during T cell development togenerate rearranged human TCRδ variable region genes in the T cells ofthe non-human organism. In some embodiments, the non-human animalprovided herein has a greater percentage of V-D and/or D-J TCRδjunctions containing non-template additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-JTCRδ junctions containing non-template additions in the geneticallymodified non-human animals provided herein is greater than percentage ofV-D and/or D-J TCRδ junctions in the corresponding non-human animal byat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%.In some embodiments, the non-human animal provided herein has a lowerpercentage of V-D TCRδ junctions not containing non-template additionsthan a corresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D and/or D-J TCRδ junctions not containing non-templateadditions in the genetically modified non-human animals provided hereinis less than percentage of V-D and/or D-J TCRδ junctions in thecorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, the non-humananimal provided herein has a greater percentage of V-D and/or D-J TCRδjunctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-JTCRδ junctions containing at least 1 N-addition in the geneticallymodified non-human animals provided herein is greater than percentage ofV-D and/or D-J TCRδ junctions in the corresponding non-human animal byat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%.In some embodiments, the non-human animal provided herein has a greaterpercentage of V-D and/or D-J TCRδ junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D and/or D-J TCRδ junctions containingat least 2 N-additions in the genetically modified non-human animalsprovided herein is greater than percentage of V-D and/or D-J TCRδjunctions in the corresponding non-human animal by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments,the non-human animal provided herein has a greater percentage of V-Dand/or D-J TCRδ junctions containing at least 3 N-additions than acorresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D and/or D-J TCRδ junctions containing at least 3N-additions in the genetically modified non-human animals providedherein is greater than percentage of V-D and/or D-J TCRδ junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D and/orD-J TCRδ junctions containing at least 4 N-additions than acorresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-D and/or D-J TCRδ junctions containing at least 4N-additions in the genetically modified non-human animals providedherein is greater than percentage of V-D and/or D-J TCRδ junctions inthe corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, at least10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-D and/or D-J TCRδ junctionsin the animal comprise non-template additions. In some embodiments, thenon-human animal has a greater frequency of unique TCRδ CDR3 sequencesthen a corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%,70%, 80%, 90% or 100%.

In some embodiments, the TCR variable region contains unrearranged humanTCRγ variable region gene segments. In some embodiments, the human TCRγvariable region gene segments rearrange during T cell development togenerate rearranged human TCRγ variable region genes in the T cells ofthe non-human organism. In some embodiments, the non-human animalprovided herein has a greater percentage of V-J TCRγ junctionscontaining non-template additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of V-J TCRγ junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J TCRγ junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a lower percentage of V-D TCRγjunctions not containing non-template additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-J TCRγjunctions not containing non-template additions in the geneticallymodified non-human animals provided herein is less than percentage ofV-J TCRγ junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J TCRγ junctions containing at least 1 N-addition than acorresponding non-human animal that does not have a nucleic acidencoding an exogenous TdT in its genome. In some embodiments, thepercentage of V-J TCRγ junctions containing at least 1 N-addition in thegenetically modified non-human animals provided herein is greater thanpercentage of V-J TCRγ junctions in the corresponding non-human animalby at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or40%. In some embodiments, the non-human animal provided herein has agreater percentage of V-J TCRγ junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J TCRγ junctions containing at least 2N-additions in the genetically modified non-human animals providedherein is greater than percentage of V-J TCRγ junctions in thecorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, the non-humananimal provided herein has a greater percentage of V-J TCRγ junctionscontaining at least 3 N-additions than a corresponding non-human animalthat does not have a nucleic acid encoding an exogenous TdT in itsgenome. In some embodiments, the percentage of V-J TCRγ junctionscontaining at least 3 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J TCRγ junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-J TCRγjunctions containing at least 4 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-J TCRγjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-J TCRγjunctions in the corresponding non-human animal by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments,at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-J TCRγ junctionsin the animal comprise non-template additions. In some embodiments, thenon-human animal has a greater frequency of unique TCRγ CDR3 sequencesthen a corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%,70%, 80%, 90% or 100%.

In some embodiments, the TCR variable region comprising unrearrangedhuman TCR variable region gene segments also includes human TCR variableregion intergenic sequences. In some embodiments, the TCR variableregion includes non-human (e.g., rodent, rat, mouse) TCR variable regionintergenic sequences. In some embodiments, the intergenic sequences areof endogenous species origin.

In some embodiments, TCR variable region comprising human variableregion gene segments and the TCR constant region gene to which thevariable region gene segments are operably linked are located at anendogenous TCRδ locus. In some embodiments, the endogenous TCR locus isan endogenous TCRα locus. In some embodiments, the endogenous TCR locusis an endogenous TCRβ locus. In some embodiments, the endogenous TCRlocus is an endogenous TCRγ locus. In some embodiments, the endogenousTCR locus is an endogenous TCRδ locus. In some embodiments, the constantregion gene to which the human variable region gene segments areoperably linked is an endogenous constant region gene, e.g., thecorresponding endogenous constant region.

In some embodiments, one or more of the endogenous TCR loci or a portionof the one or more endogenous loci (e.g., a variable region and/or aconstant region) in the genome of the non-human animal provided hereinis inactivated. Endogenous TCR variable region gene loci and portionsthereof can be inactivated using any method known in the art, including,but not limited to, the deletion of the locus or a portion thereof fromthe genome of the organism, the replacement of a locus or a portionthereof with a different nucleic acid sequence, the inversion of aportion of the locus and/or the displacement of a portion of the locusto another position in the genome of the non-human organism. In someembodiments the inactivation of the locus is only a partialinactivation. In some embodiments, the variable region of the locus isinactivated but the constant region remains functional (e.g., because itis operably linked to non-endogenous variable region gene segments).Examples of inactivated TCR loci are described, for example, inMombaerts et al., Proc. Natl. Acad. Sci. USA 88:3084-3087 (1991) andMombaerts et al., Nature 390:225-231 (1992), each of which is herebyincorporated by reference.

In some embodiments, the genetically modified non-human animal providedherein expresses TCR having human variable domains (e.g., a humanvariable domain derived from the unrearranged human variable region genesegments described herein). In some embodiments, the human variabledomain is a human TCRα variable domain. In some embodiments, the humanvariable domain is a human TCRβ variable domain. In some embodiments,the human variable domain is a human TCRγ variable domain. In someembodiments, the human variable domain is a human TCRδ variable domain.In some embodiments, the TCR produced by the non-human animals have bothhuman TCRα variable domains and human TCRβ variable domains. In someembodiments, the TCR produced by the non-human animals have both humanTCRγ variable domains and human TCRδ variable domains. In someembodiments, the TCR produced by the non-human animals have both humanTCRα variable domains and human TCRβ variable domains, and both humanTCRγ variable domains and human TCRδ variable domains. In someembodiments, the TCR have human constant domains. In some embodiments,the constant domains are of non-human origin. For example, in someembodiments, constant domains are of endogenous species origin. In someembodiments, the constant domains are of mouse or rat origin.

Non-human Animals Expressing Chimeric Antigen Receptors (CARs) andExogenous TdT

In certain aspects, provided herein are genetically modified non-humananimals and non-human animal ES cells comprising exogenous TdT asdescribed herein that also comprise chimeric antigen receptor (CAR)loci. Such CAR loci generally comprise a variable region and a constantregion. The variable region includes unrearranged human Ig variableregion gene segments, while the constant region locus includes a TCRconstant region gene, wherein the Ig variable region gene segments areoperably linked to the constant region gene. In some embodiments, theTCR constant region gene is a non-human TCR constant region gene. Insome embodiments, the TCR constant region gene is a rodent constantregion gene, such as a rat constant region gene or a mouse constantregion gene. In some embodiments, the constant region gene is ofendogenous species origin. In some embodiments, the TCR constant regiongene is a human constant region gene.

In some embodiments, the CAR loci described herein are located at anendogenous TCR loci. For example, in some embodiments, a CAR locuscomprising a TCRα constant region gene is located at an endogenous TCRαconstant region locus. In some embodiments, such a locus is created byreplacing some or all of the TCRα unrearranged variable region with anunrearranged Ig variable region. In some embodiments, a CAR locuscomprising a TCRβ constant region gene is located at an endogenous TCRβconstant region locus. In some embodiments, such a locus is created byreplacing some or all of the TCRβ unrearranged variable region with anunrearranged Ig variable region.

In certain embodiments, the CAR variable region locus will containunrearranged human Ig variable region gene segments. Exemplary variableregion loci comprising human variable region gene segments have beendescribed in the art. For example, such loci are described in U.S. Pat.Nos. 5,633,425, 5,770,429, 5,814,318, 6,075,181, 6,114,598, 6,150,584,6,998,514, 7,795,494, 7,910,798, 8,232,449, 8,502,018, 8,697,940,8,703,485, 8,754,287, 8,791,323, 8,907,157, 9,035,128, 9,145,588, and9,206,263 and each of which is hereby incorporated by reference in itsentirety, as well as in U.S. Pat. Pub. Nos. 2008/0098490, 2010/0146647,2011/0195454, 2012/0167237, 2013/0145484, 2013/0167256, 2013/0219535,2013/0326647, 2014/013275, 2014/093908, 2015/0113668 and 2016/0081314,each of which is hereby incorporated by reference in its entirety, andin PCT Pub. Nos. WO2007117410, WO2008151081, WO2009157771, WO2010039900,WO2011004192, WO2011123708, WO2014093908 and WO2016/044745, each ofwhich are hereby incorporated by reference in its entirety.

In certain embodiments, the CAR variable region locus containsunrearranged human Ig heavy chain variable region gene segments. In someembodiments, the unrearranged human Ig variable region gene segmentscomprise a plurality of human V_(H) segments, one or more human D_(H)segments and one or more human Ju segments. In some embodiments, theunrearranged human Ig variable region gene segments comprise at least 3V_(H) gene segments, at least 18 V_(H) gene segments, at least 20 V_(H)gene segments, at least 30 V_(H) gene segments, at least 40 V_(H) genesegments, at least 50 V_(H) gene segments, at least 60 V_(H) genesegments, at least 70 V_(H) gene segments, or at least 80 V_(H) genesegments. In some embodiments, the unrearranged human Ig gene segmentsinclude all of the human D_(H) gene segments. In some embodiments, theCAR variable region further comprises TCRβ variable region gene segments(e.g., V, D and/or J gene segments). In one embodiment, the CAR variableregion further comprises distal TCR Vβ gene segments, e.g., TCR Vβ31gene segment. In another embodiment, the distal TCR Vβ gene segments,e.g., TCR Vβ31 gene segment, has been functionally inactivated ordeleted. In some embodiments, the unrearranged human Ig gene segmentsinclude all of the human J_(H) gene segments. Exemplary variable regionscomprising Ig heavy chain gene segments are provided, for example, inMacdonald et al., Proc. Natl. Acad. Sci. USA 111:5147-52 andsupplemental information, which is hereby incorporated by reference.

In some embodiments, the human immunoglobulin heavy chain variableregion gene segments rearrange during T cell development to generaterearranged human heavy chain variable region genes in the T cells of thenon-human organism. In some embodiments, the non-human animal providedherein has a greater percentage of V-D and/or D-J immunoglobulin heavychain junctions containing non-template additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-Jimmunoglobulin heavy chain junctions containing non-template additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-D and/or D-J immunoglobulin heavy chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a lower percentage of V-Dimmunoglobulin heavy chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D and/or D-J immunoglobulin heavy chainjunctions not containing non-template additions in the geneticallymodified non-human animals provided herein is less than percentage ofV-D and/or D-J immunoglobulin heavy chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of V-D and/or D-J immunoglobulin heavychain junctions containing at least 1 N-addition than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-Jimmunoglobulin heavy chain junctions containing at least 1 N-addition inthe genetically modified non-human animals provided herein is greaterthan percentage of V-D and/or D-J immunoglobulin heavy chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D and/orD-J immunoglobulin heavy chain junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D and/or D-J immunoglobulin heavy chainjunctions containing at least 2 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-Dand/or D-J immunoglobulin heavy chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, the non-human animal providedherein has a greater percentage of V-D and/or D-J immunoglobulin heavychain junctions containing at least 3 N-additions than a correspondingnon-human animal that does not have a nucleic acid encoding an exogenousTdT in its genome. In some embodiments, the percentage of V-D and/or D-Jimmunoglobulin heavy chain junctions containing at least 3 N-additionsin the genetically modified non-human animals provided herein is greaterthan percentage of V-D and/or D-J immunoglobulin heavy chain junctionsin the corresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In some embodiments, thenon-human animal provided herein has a greater percentage of V-D and/orD-J immunoglobulin heavy chain junctions containing at least 4N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-D and/or D-J immunoglobulin heavy chainjunctions containing at least 4 N-additions in the genetically modifiednon-human animals provided herein is greater than percentage of V-Dand/or D-J immunoglobulin heavy chain junctions in the correspondingnon-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 30% or 40%. In some embodiments, at least 10%, 15%, 20%, 25%,30%, 35% or 40% of the V-D and/or D-J immunoglobulin heavy chainjunctions in the animal comprise non-template additions. In someembodiments, the non-human animal has a greater frequency of uniqueimmunoglobulin heavy chain CDR3 sequences then a corresponding non-humananimal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100%.

In some embodiments, the CAR variable gene locus comprising unrearrangedhuman Ig heavy chain variable region gene segments also includes humanIg heavy chain variable region intergenic sequences. In someembodiments, the CAR variable gene locus includes non-human (e.g.,rodent, rat, mouse) Ig heavy chain variable region intergenic sequences.In some embodiments, the CAR variable gene locus includes human ornon-human (e.g., rodent, rat, mouse) TCRβ variable region intergenicsequences. For example, in some embodiments the unrearranged variableregion of the CAR locus comprises one or more (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) trypsinogen (TRY)genes (e.g., TRY genes and/or pseudogenes normally present in the TCR(3variable region locus). In some embodiments, the TRY genes arenon-human, e.g., mouse TRY genes. In some embodiments, the mouse TRYgenes are selected from the group consisting of Try1, Try2, Try3, Try4,TryS, Try6, Try7, Try8, Try9, Try10, Try11, Try12, Try13, Try14, Try15,Try16, Try17, Try18, Try19 and Try20. In some embodiments, one or moreTRY genes are located upstream of the V_(H) segments of the unrearrangedvariable region. In some embodiments, one or more TRY genes are locateddownstream of the V_(H) segments and upstream of the D_(H) segments ofthe unrearranged variable region. In some embodiments, Try1-7 arelocated upstream of the V_(H) segments of the unrearranged variableregion and Try 8-20 are located downstream of the V_(H) segments andupstream of the D_(H) segments of the unrearranged variable region.Additional information regarding the TRY genes located in the humanand/or mouse TCRβ locus is provided in Glusman et al., Immunity15:337-349 (2001) and Skok et al., Nature Immunology 8:378-387 (2007),each of which is incorporated by reference. In some embodiments, the CARgene locus comprises non-human regulatory elements (e.g., non-humanpromoters and/or enhancers). In some embodiments, the non-humanregulatory elements are rodent regulatory elements (e.g., rat or mousepromoters or enhancers). In some embodiments, the CAR locus comprises anIgM enhancer (Eμ). In some embodiments, the IgM enhancer is a non-humanEμ (e.g., a rodent Eμ, such as a mouse or rat Eμ).

In certain embodiments, the CAR variable region locus containsunrearranged human Ig κ variable region gene segments. In someembodiments, the unrearranged human immunoglobulin variable region genesegments comprise a plurality of human V_(κ) segments and one or morehuman J_(κ) segments. In some embodiments, the immunoglobulin variableregion gene segments comprise four functional V_(κ) segments and allhuman J_(κ) segments. In some embodiments, the immunoglobulin variableregion gene segments comprise 16 functional V_(κ) segments and all humanJ_(κ) segments. In some embodiments, the unrearranged humanimmunoglobulin variable region gene segments comprise all of the humanV_(κ) segments and all human J_(κ) segments (e.g., all functional humanV_(κ) segments and J_(κ) segments). Exemplary variable regionscomprising Ig κ gene segments are provided, for example, in Macdonald etal., Proc. Natl. Acad. Sci. USA 111:5147-52 and supplementalinformation, which is hereby incorporated by reference. In someembodiments, the unrearranged human immunoglobulin variable region genesegments comprise all of the human J_(κ) segments. In some embodiments,the CAR variable region further comprises TCRα variable region genesegments (e.g., V, and/or J gene segments).

In some embodiments, the human immunoglobulin κ variable region genesegments rearrange during T cell development to generate rearrangedhuman κ variable region genes in the T cells of the non-human organism.In some embodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a lower percentageof V-J immunoglobulin κ chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctions notcontaining non-template additions in the genetically modified non-humananimals provided herein is less than percentage of V-J immunoglobulin κchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 1N-addition than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 1 N-addition in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 2 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 3N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 3 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin κ chain junctions containing at least 4N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin κ chain junctionscontaining at least 4 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinκ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-Jimmunoglobulin κ chain junctions in the animal comprise non-templateadditions. In some embodiments, the non-human animal has a greaterfrequency of unique immunoglobulin κ chain CDR3 sequences then acorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%,90% or 100%. In some embodiments, the non-human animal provided hereinhas at least 900, 1000, 1100, 1200, 1300, 1400, 1500 or 1700 uniqueimmunoglobulin κ chain CDR3 sequences per 10,000 immunoglobulin κ chainCDR3 sequences.

In certain embodiments, the CAR variable region locus containsunrearranged human Ig λ variable region gene segments. In someembodiments, the unrearranged human immunoglobulin variable region genesegments comprise a plurality of human V_(λ) segments and one or morehuman J_(λ) segments. In some embodiments, the unrearranged humanimmunoglobulin variable region gene segments comprise all of the humanV_(λ) segments (e.g., all functional human V_(λ) segments). In someembodiments, the unrearranged human immunoglobulin variable region genesegments comprise all of the human J_(λ) segments. In some embodiments,the CAR variable region further comprises TCRα variable region genesegments (e.g., V, and/or J gene segments). Exemplary variable regionscomprising Ig λ gene segments are provided, for example, U.S. Pat. Pub.Nos. 2012/0073004 and 2002/0088016, each of which is hereby incorporatedby reference.

In some embodiments, the human immunoglobulin λ variable region genesegments rearrange during T cell development to generate rearrangedhuman λ variable region genes in the T cells of the non-human organism.In some embodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containingnon-template additions than a corresponding non-human animal that doesnot have a nucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining non-template additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a lower percentageof V-J immunoglobulin λ chain junctions not containing non-templateadditions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctions notcontaining non-template additions in the genetically modified non-humananimals provided herein is less than percentage of V-J immunoglobulin λchain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 1N-addition than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 1 N-addition in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 2N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 2 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 3N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 3 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, the non-human animal provided herein has a greaterpercentage of V-J immunoglobulin λ chain junctions containing at least 4N-additions than a corresponding non-human animal that does not have anucleic acid encoding an exogenous TdT in its genome. In someembodiments, the percentage of V-J immunoglobulin λ chain junctionscontaining at least 4 N-additions in the genetically modified non-humananimals provided herein is greater than percentage of V-J immunoglobulinλ chain junctions in the corresponding non-human animal by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 40%. In someembodiments, at least 10%, 15%, 20%, 25%, 30%, 35% or 40% of the V-Jimmunoglobulin λ chain junctions in the animal comprise non-templateadditions. In some embodiments, the non-human animal has a greaterfrequency of unique immunoglobulin λ chain CDR3 sequences then acorresponding non-human animal by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 40%, 45%, 50%, 60%, 70%, 80%,90% or 100%. In some embodiments, the non-human animal provided hereinhas at least 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290 or 300 unique immunoglobulin λ chain CDR3 sequences per10,000 immunoglobulin λ chain CDR3 sequences.

In some embodiments, the CAR variable gene locus containing unrearrangedhuman Ig light chain variable region gene segments also includes humanIg light chain variable region intergenic sequences (e.g., κ variableregion intergenic sequences and/or λ variable region intergenicsequences). In some embodiments, the CAR variable gene locus includesnon-human (e.g., rodent, rat, mouse) Ig light chain variable regionintergenic sequences (e.g., κ variable region intergenic sequencesand/or λ variable region intergenic sequences). In some embodiments, theCAR variable gene locus includes human or non-human (e.g., rodent, rat,mouse) TCRα variable region intergenic sequences. In some embodiments,the CAR gene locus comprises non-human regulatory elements (e.g.,non-human promoters and/or enhancers). In some embodiments, thenon-human regulatory elements are rodent regulatory elements (e.g., rator mouse promoters or enhancers).

In some embodiments, the CAR variable region locus is a rearrangedvariable region locus comprising a Ig heavy chain variable region gene(a universal heavy chain variable region). In some embodiments, therearranged Ig heavy chain variable region gene is a human rearranged Igheavy chain variable region gene. Use of universal heavy chain variableregions facilitate the generation of bispecific antibodies in which atleast one antigen-binding domain has specificity for a peptide/MHCcomplex. Exemplary rearranged Ig heavy chain variable regions areprovided in U.S. Patent Pub. No. 2014/0245468, which is herebyincorporated by reference.

In some embodiments, the CAR variable region locus is a rearrangedvariable region locus comprising a Ig light chain variable region gene(a universal light chain variable region). In some embodiments, therearranged Ig light chain variable region gene is a human rearranged Iglight chain variable region gene. Use of universal light chain variableregions facilitate the generation of bispecific antibodies in which atleast one antigen-binding domain has binding specificity for apeptide/MHC complex. Exemplary rearranged Ig heavy chain variableregions are provided in U.S. Patent Pub. No. 2013/0185821, which ishereby incorporated by reference.

Other Genetic Modifications

In some embodiments, the genetically modified non-human animals and EScells described herein that express exogenous TdT, humanized TCRs orCARs also express and/or comprise in their genome loci encodinghumanized MHC class I a chain polypeptides (e.g., humanized HLA-A,HLA-B, HLA-C, HLA-E, HLA-F, HLA-g, HLA-K and/or HLA-L). In someembodiments, the humanized MHC class I α chain polypeptide is fullyhuman. In some embodiments, the humanized MHC class I α chainpolypeptide comprises a human extracellular domain (e.g., a human α1,α2, and α3 domains) and a cytoplasmic domain of endogenous speciesorigin. Humanized MHC class I a chain polypeptides, loci encodinghumanized MHC class I α chain polypeptides and non-human animalsexpressing humanized MHC class I α chain polypeptides are described inU.S. Pat. Pub. Nos. 2013/0111617, 2013/0185819 and 2014/0245467, each ofwhich is incorporated by reference herein.

In some embodiments, the genetically modified non-human animals and EScells described herein that express exogenous TdT, humanized TCRs orCARs also express and/or comprise in their genome a locus encodinghumanized β-2-microglobulin polypeptide. Humanized β-2-microglobulinpolypeptides, loci encoding humanized β-2-microglobulin polypeptides andnon-human animals expressing humanized β-2-microglobulin polypeptidesare described in U.S. Pat. Pub. Nos. 2013/0111617 and 2013/0185819, eachof which is incorporated by reference herein.

In some embodiments, the genetically modified non-human animals and EScells described herein that express exogenous TdT, humanized TCRs orCARs also express and/or comprise in their genome a loci encodinghumanized MHC class II α chain polypeptides (e.g., humanized HLA-DMA,HLA-DOA, HLA-DPA, HLA-DQA and/or HLA-DRA). In some embodiments, thehumanized MHC class II α chain polypeptide is fully human. In someembodiments, the humanized MHC class II α chain polypeptide comprises ahuman extracellular domain and a cytoplasmic domain of endogenousspecies origin. Humanized MHC class II α chain polypeptides, lociencoding humanized MHC class II α chain polypeptides and non-humananimals expressing humanized MHC class II α chain polypeptides aredescribed in U.S. Pat. Nos. 8,847,005 and 9,043,996 and U.S. Pat. Pub.No. 2014/0245467, each of which is incorporated by reference herein.

In some embodiments, the genetically modified non-human animals and EScells described herein that express exogenous TdT, humanized TCRs orCARs also express and/or comprise in their genome a loci encodinghumanized MHC class II β chain polypeptides (e.g., humanized HLA-DMB,HLA-DOB, HLA-DPB, HLA-DQB and/or HLA-DRB). In some embodiments, thehumanized MHC class II β chain polypeptide is fully human. In someembodiments, the humanized MHC class II β chain polypeptide comprises ahuman extracellular domain and a cytoplasmic domain of endogenousspecies origin. Humanized MHC class II β chain polypeptides, lociencoding humanized MHC class II β chain polypeptides and non-humananimals expressing humanized MHC class II β chain polypeptides aredescribed in U.S. Pat. Nos. 8,847,005 and 9,043,996 and U.S. Pat. Pub.No. 2014/0245467, each of which is incorporated by reference herein.

Genetically modified non-human animals comprising exogenous TdT,humanized TCR loci and humanized MHC I and/or MHC II (MHC IIα/IIβ) locican be generated by breeding using conventional methods; alternatively,they can be generated by homologous recombination in ES cells alreadycomprising one or more genetically engineered loci (e.g., humanized TCRloci), and generating a non-human animal from said ES cells.

Genetically modified non-human animals comprising exogenous TdT,humanized CAR loci and humanized MHC I and/or MHC II (MHC IIα/IIβ) locican be generated by breeding using conventional methods; alternatively,they can be generated by homologous recombination in ES cells alreadycomprising one or more genetically engineered loci (e.g., humanized CARloci), and generating a non-human animal from said ES cells.

In some embodiments, the genetically modified non-human animals and EScells described herein that express exogenous TdT, humanized TCRs orCARs also express and/or comprise in their genome a locus encoding ahumanized CD8 α chain polypeptide. In some embodiments, the humanizedCD8 α chain polypeptide is fully human. In some embodiments, thehumanized CD8 α chain polypeptide comprises a human extracellularimmunoglobulin domain and a cytoplasmic domain of endogenous speciesorigin. Humanized CD8 α chain polypeptides, loci encoding humanized CD8α chain polypeptides and non-human animals expressing humanized CD8 αchain polypeptides are described in U.S. Pat. Pub. Nos. 2014/0245466which is incorporated by reference herein.

In some embodiments, the genetically modified non-human animals and EScells described herein that express exogenous TdT, humanized TCRs orCARs also express and/or comprise in their genome a locus encoding ahumanized CD8 β chain polypeptide. In some embodiments, the humanizedCD8 β chain polypeptide is fully human. In some embodiments, thehumanized CD8 β chain polypeptide comprises a human extracellularimmunoglobulin domain and a cytoplasmic domain of endogenous speciesorigin. Humanized CD8 β chain polypeptides, loci encoding humanized CD8β chain polypeptides and non-human animals expressing humanized CD8 βchain polypeptides are described in U.S. Pat. Pub. Nos. 2014/0245466which is incorporated by reference herein.

In some embodiments, the genetically modified non-human animals and EScells described herein that express exogenous TdT, humanized TCRs orCARs also express and/or comprise in their genome a locus encoding ahumanized CD4 polypeptide. In some embodiments, the humanized CD4polypeptide is fully human. In some embodiments, the humanized CD4polypeptide comprises at least one human extracellular immunoglobulindomain and a cytoplasmic domain of endogenous species origin. In someembodiments, the humanized CD4 polypeptide comprises at least a human D1immunoglobulin domain, a human D2 immunoglobulin domain, and a human D3immunoglobulin domain, and a cytoplasmic domain of endogenous speciesorigin. In some embodiments, the humanized CD4 polypeptide comprises ahuman D1 immunoglobulin domain, a human D2 immunoglobulin domain, ahuman D3 immunoglobulin domain, a D4 immunoglobulin domain of endogenousspecies origin and a cytoplasmic domain of endogenous species origin.Humanized CD4 polypeptides, loci encoding humanized CD4 polypeptides andnon-human animals expressing humanized CD4 polypeptides are described inU.S. Pat. Pub. Nos. 2014/0245466 which is incorporated by referenceherein.

Genetically modified non-human animals comprising exogenous TdT,humanized TCR loci and humanized CD4 and/or CD8 (CD8α/CD8β) loci can begenerated by breeding using conventional methods; alternatively, theycan be generated by homologous recombination in ES cells alreadycomprising one or more genetically engineered loci (e.g., humanized TCRloci), and generating a non-human animal from said ES cells.

Genetically modified non-human animals comprising exogenous TdT,humanized CAR loci and humanized CD4 and/or CD8 (CD8α/CD8β) loci can begenerated by breeding using conventional methods; alternatively, theycan be generated by homologous recombination in ES cells alreadycomprising one or more genetically engineered loci (e.g., humanized CARloci), and generating a non-human animal from said ES cells.

Methods of Using the Genetically Modified Non-Human Animals

In certain aspects, provided herein are methods of using the geneticallymodified non-human animals described herein to generate antigen bindingproteins (e.g., antibodies, CARs, TCRs), cells expressing such antigenbinding proteins (e.g., B cells, T cells, B cell hybridomas, T cellhybridomas) and nucleic acids encoding such antigen binding proteins orportions thereof (e.g., variable domains). In some embodiments, providedherein are methods of making more diverse antigen binding proteins(e.g., antibodies, CARs, TCRs). In some embodiments, provided herein aremethods of making rearranged variable regions of antigen bindingproteins (e.g., antibodies, CARs, TCRs) that have increased numbers ofnucleotide additions.

In certain embodiments, the method comprises exposing a geneticallymodified non-human animal described herein that has been modified toexpress exogenous TdT and antibodies or antigen-binding fragmentsthereof having human variable domains to an antigen such that thegenetically modified non-human animal produces an antibody or anantigen-binding fragment thereof comprising a human variable domainspecific for the antigen.

In some embodiments the method comprises exposing a genetically modifiednon-human animal described herein that has been modified to expressexogenous TdT and antibodies or antigen-binding fragments thereof havinghuman variable domains to an antigen; and obtaining a B cell expressingan antibody or an antigen-binding fragment thereof comprising a humanvariable domain specific for the antigen from the non-human animal.

In some embodiments, the method comprises exposing a geneticallymodified non-human animal described herein that has been modified toexpress exogenous TdT and antibodies or antigen-binding fragmentsthereof having human variable domains to an antigen; obtaining a B cellexpressing an antibody or an antigen-binding fragment thereof comprisinga human variable domain specific for the antigen from the non-humananimal; and making a hybridoma from the B cell.

In some embodiments, the method comprises exposing a geneticallymodified non-human animal described herein that has been modified toexpress exogenous TdT and antibodies or antigen-binding fragmentsthereof having human variable domains to an antigen; and obtaining anucleic acid encoding a human immunoglobulin variable domain specificfor the antigen from the non-human animal.

In certain embodiment, the method comprises exposing a geneticallymodified non-human animal described herein that has been modified toexpress exogenous TdT and antibodies or antigen-binding fragmentsthereof having human variable domains to an antigen; obtaining a B cellexpressing an antibody or an antigen-binding fragment thereof comprisinga human variable domain specific for the antigen from the non-humananimal; optionally making a hybridoma from the B cell; and obtaining anucleic acid encoding a human immunoglobulin variable domain specificfor the antigen from the B cell or the hybridoma.

In some embodiments, the method comprises exposing a non-human animaldescribed herein that has been modified to express exogenous TdT andantibodies or antigen-binding fragments thereof having human variabledomains to an antigen; obtaining a B cell expressing an antibody or anantigen-binding fragment thereof comprising a human variable domainspecific for the antigen from the non-human animal; optionally making ahybridoma from the B cell; obtaining a nucleic acid encoding a humanimmunoglobulin variable domain specific for the antigen from the B cellor the hybridoma; operably linking the nucleic acid encoding theimmunoglobulin variable domain with a nucleic acid encoding a humanimmunoglobulin constant domain in a host cell; and culturing the hostcell under conditions such that the host cell expresses a human antibodycomprising the immunoglobulin variable domain and the immunoglobulinconstant domain.

In some embodiments, the method comprises exposing a geneticallymodified non-human animal described herein that has been modified toexpress exogenous TdT and TCR having human variable domains to anantigen comprising a peptide or a nucleic acid encoding an antigencomprising a peptide such that the peptide is presented on a MHC in thenon-human animal; and obtaining a T cell expressing a TCR specific forthe peptide presented on the MHC from the genetically modified non-humananimal.

In some embodiments, the method comprises exposing a geneticallymodified non-human animal described herein that has been modified toexpress exogenous TdT and TCR having human variable domains to anantigen comprising a peptide or a nucleic acid encoding an antigencomprising a peptide such that the peptide is presented on a MHC in thenon-human animal; obtaining a T cell expressing a TCR specific for thepeptide presented on the MHC from the genetically modified non-humananimal; and making a T cell hybridoma from the T cell.

In some embodiments, the method comprises exposing a non-human animaldescribed herein that has been modified to express exogenous TdT and TCRhaving human variable domains to an antigen comprising a peptide or anucleic acid encoding an antigen comprising a peptide such that thepeptide is presented on a MHC in the non-human animal; obtaining a Tcell expressing a TCR specific for the peptide presented on the MHC fromthe genetically modified non-human animal; and isolating a nucleic acidencoding a human TCR variable domain of the TCR from the T cell.

In some embodiments, the method comprises exposing a non-human animaldescribed herein that has been modified to express exogenous TdT and TCRhaving human variable domains to an antigen comprising a peptide or anucleic acid encoding an antigen comprising a peptide such that thepeptide is presented on a MHC in the non-human animal; obtaining a Tcell expressing a TCR specific for the peptide presented on the MHC fromthe genetically modified non-human animal; isolating a nucleic acidencoding a TCR variable domain of the TCR from the T cell; and operablylinking the nucleic acid encoding the TCR variable domain with a TCRconstant domain in a cell such that the cell expresses a TCR comprisingthe TCR variable domain and the TCR constant domain.

In some embodiments, the method comprises exposing a geneticallymodified non-human animal described herein that has been modified toexpress exogenous TdT and CAR having human variable domains to anantigen comprising a peptide or a nucleic acid encoding an antigencomprising a peptide such that the peptide is presented on a MHC in thenon-human animal; and obtaining a T cell expressing a CAR specific forthe peptide presented on the MHC from the genetically modified non-humananimal.

In some embodiments, the method comprises exposing a geneticallymodified non-human animal described herein that has been modified toexpress exogenous TdT and CAR having human variable domains to anantigen comprising a peptide or a nucleic acid encoding an antigencomprising a peptide such that the peptide is presented on a MHC in thenon-human animal; obtaining a T cell expressing a CAR specific for thepeptide presented on the MHC from the genetically modified non-humananimal; and making a T cell hybridoma from the T cell.

In some embodiments, the method comprises exposing a non-human animaldescribed herein that has been modified to express exogenous TdT and CARhaving human variable domains to an antigen comprising a peptide or anucleic acid encoding an antigen comprising a peptide such that thepeptide is presented on a MHC in the non-human animal; obtaining a Tcell expressing a chimeric antigen receptor (CAR) specific for thepeptide presented on the MHC from the genetically modified non-humananimal; and isolating a nucleic acid encoding a human TCR variabledomain of the CAR from the T cell.

In some embodiments, the method comprises exposing a non-human animaldescribed herein that has been modified to express exogenous TdT and CARhaving human variable domains to an antigen comprising a peptide or anucleic acid encoding an antigen comprising a peptide such that thepeptide is presented on a MHC in the non-human animal; obtaining a Tcell expressing a chimeric antigen receptor (CAR) specific for thepeptide presented on the MHC from the genetically modified non-humananimal; isolating a nucleic acid encoding a human immunoglobulinvariable domain of the CAR from the T cell; and operably linking thenucleic acid encoding the human immunoglobulin variable domain with ahuman immunoglobulin constant domain in a cell such that the cellexpresses an antibody comprising the human immunoglobulin variabledomain and the human immunoglobulin constant domain.

In certain embodiments, the methods described herein include a step inwhich a non-human animal described herein is exposed to an antigen(immunized) in order to induce an immune response (e.g., a B cell immuneresponse and/or a T cell immune response). In some embodiments thegenetically modified non-human animal is immunized with a whole proteinantigen or a fragment thereof. Rodents can be immunized by any methodknown in the art (see, for example, Harlow and Lane (1988) Antibodies: ALaboratory Manual 1988 Cold Spring Harbor Laboratory; Malik and Lillehoj(1994) Antibody Techniques, Academic Press, CA).

In some embodiments, the genetically modified non-human animal isexposed to the antigen by administering to the non-human animal a virus(e.g., a retrovirus, an adenovirus, a vaccinia virus or a lentivirus)comprising a nucleic acid sequence encoding the antigen. Methods forviral vaccination are provided, for example, in U.S. Pat. Nos.6,001,349, 8,663,622, 8,691,502, 8,377,688, as well as Precopio et al.,JEM 204:1405-1416 (2007), each of which is hereby incorporated byreference in its entirety. In some embodiments, the non-human animal isadministered the virus directly. In some embodiments, a cell (e.g., anantigen presenting cell, such as a dendritic cell) is infected with thevirus in vitro or ex vivo which is then administered to the non-humananimal. In some embodiments, the virus encodes a peptide/MHC complex(e.g., a single-chain peptide/MHC complex). Examples of single-chainpeptide/MHC based vaccines are provided in Truscott et al., J. Immunol.178:6280-6289 (2007), EP1773383, Kim et al., Vaccine 30:2178-2186(2012), Kim et al., J. Immunol. 184:4423-4430 (2010), each of which arehereby incorporated by reference.

In some embodiments, the genetically modified non-human animal isexposed to the antigen by administering to the animal a nucleic acidencoding the antigen. In some embodiments, the non-human animal isadministered a nucleic acid encoding a single chain peptide/MHC complex.Examples of single-chain peptide/MHC based vaccines are provided inTruscott et al., J. Immunol. 178:6280-6289 (2007), EP1773383, Kim etal., Vaccine 30:2178-2186 (2012), Kim et al., J. Immunol. 184:4423-4430(2010), each of which are hereby incorporated by reference. In certainembodiments, the nucleic acid is a DNA vector. The delivery of nucleicacids can be by any technique known in the art including viral mediatedgene transfer and liposome mediated gene transfer. A polynucleotide ofinterest is associated with a liposome to form a gene delivery vehicleas described in, for example, U.S. Pat. Nos. 6,770,291, 7,001,614,6,749,863, 5,512,295 and 7,112,338, each of which is hereby incorporatedby reference. In some embodiments, the nucleic acid is an mRNA vector.Exemplary methods for generating and administering mRNA vectors aredescribed in, for example, U.S. Pat. No. 8,278,036 and U.S. Pat. Pub.Nos. 2013/151736 and 2012/135805, each of which is hereby incorporatedby reference.

In some embodiments, antigen is a cancer-associated antigen. Examples ofcancer-associated antigens include, but are not limited to, adipophilin,AIM-2, ALDH1A1, alpha-actinin-4, alpha-fetoprotein (“AFP”), ALK,ANKRD30A, ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2,beta-catenin, BING-4, BIRC7, CA-125, CA9, CALCA, carcinoembryonicantigen (“CEA”), CALR, CASP-5, CASP-8, CCRS, CD19, CD20, CD22, CD27,CD274, CD30, CD33, CD38, CD40, CD44, CD45, CD52, CD56, CD79, Cdc27,CDK12, CDK4, CDKN2A, CEA, CLEC12A, CLPP, COA-1, CPSF, CSNK1A1, CTAG1,CTAG2, cyclin D1, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, EGFR,EGFR variant III, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM,EphA2, EphA3, epithelial tumor antigen (“ETA”), ERBB3, ERBB4, ETV6-AML1fusion protein, EZH2, FCRL3, FGFS, FLT3-ITD, FN1, FOLR1, G250/MN/CAIX,GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gp100/Pmel17, GPNMB,GM3, GPR112, IL3RA, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11,HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A, KIT, KK-LC-1, KKLC1, KMHN1 alsoknown as CCDC110, KRAS, LAGE-1, LDLR-fucosyltransferaseAS fusionprotein, Lengsin, LGRS, LMP2, M-CSF, MAGE-A1, MAGE-A10, MAGE-Al2,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malicenzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, MELMelan-A/MART-1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC2, MUC3, MUC4,MUCS, MUCSAC, MUC16, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I,N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OAL OGT, OS-9,OX40, P polypeptide, p53, PAP, PAX3, PAXS, PBF, PLAC1, PMEL,pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”),PPP1R3B, PRAME, PRDXS, PRLR, PSA, PSMA, PTPRK, RAB38/NY-MEL-1, RAGE-1,RBAF600, RET, RGSS, RhoC, RNF43, ROR1, RU2AS, SAGE, SART1, SART3,secemin 1, SIRT2, SLAMF7, SLC39A6, SNRPD1, SOX10, Sp17, SPA17, SSX-2,SSX-4, STEAP1, STEAP2, survivin, SYT-SSX1 or -SSX2 fusion protein,TAG-1, TAG-2, Telomerase, TERT, TGF-betaRll, Thompson-nouvelle antigen,TMPRSS2, TNFRSF17, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75,TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), UPK3A, VEGF, VTCN1,WT1, XAGE-1b/GAGED2a. In some embodiments, the antigen is a neo-antigen.

In some embodiment, the antigen is an antigen expressed by an infectiouspathogen. In some embodiments, the pathogen is a virus, a bacteria, afungus, a helminth, or a protozoa. Nonlimiting examples of virusesinclude HIV, hepatitis A, hepatitis B, hepatitis C, herpes virus (e.g.,HSV-1, HSV-2, CMV, HAV-6, VZV, Epstein Barr virus), adenovirus,influenza virus, flavivirus, echovirus, rhinovirus, coxsackie virus,coronavirus, respiratory syncytial virus, mumps virus, rotavirus,measles virus, rubella virus, parvovirus, vaccinia virus, HTLV, denguevirus, papillomavirus, molluscum virus, poliovirus, rabies virus, JCvirus, ebola virus, and arboviral encephalitis virus antigen. In someembodiments, the parasite is malaria. In some embodiments, pathogen isAspergillus, Brugia, Candida, Chlamydia, Coccidia, Cryptococcus,Dirofilaria, Gonococcus, Histoplasma, Klebsiella, Legionella,Leishmania, Meningococci, Mycobacterium, Mycoplasma, Paramecium,Pertussis, Plasmodium, Pneumococcus, Pneumocystis, Pseudomonas,Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,Toxoplasma and Vibriocholerae. Exemplary species include Neisseriagonorrhea, Mycobacterium tuberculosis, Candida albicans, Candidatropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group BStreptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granulomainguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus.Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus,Campylobacter fetus intestinalis, Leptospira pomona, Listeriamonocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas foetus,Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonellaabortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa,Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis,Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa,Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridiumbotulinum; or, a fungus, such as, e.g., Paracoccidioides brasiliensis;or other pathogen, e.g., Plasmodium falciparum.

In some embodiments of the methods described herein, the method includesthe step of obtaining a T cell and/or or B cell from the geneticallymodified non-human animal. In certain embodiments, any method known inthe art can be used to obtain such cells. For example, such T cellsand/or B cells can be obtained from the spleen, lymph nodes and/orperipheral blood of the animal. Such T cells and/or B cells can bescreened for binding specificity using methods available in the art.

In some embodiments, the methods described herein include the step ofmaking a B cell hybridoma from a B cell. Methods useful for making a Bcell hybridoma are known in the art and described, for example, inHarlow and Lane (1988) Antibodies: A Laboratory Manual 1988 Cold SpringHarbor Laboratory; Malik and Lillehoj (1994) Antibody Techniques,Academic Press, CA, which is hereby incorporated by reference.

In some embodiments, the methods described herein include the step ofmaking a T cell hybridoma from a T cell. Methods useful for making a Tcell hybridoma are known in the art and described, for example, inHedrick et al., Cell 30:141-152 (1982) and Kruisbeek Curr. Protoc.Immunol. Chapter 3 (2001) and White et al., Methods in Molecular Biology134:185-193 (2000), each of which is hereby incorporated by reference.

In some embodiments, the methods provided herein include the step ofisolating a nucleic acid encoding an Ig or TCR variable region. In someembodiments of the methods described herein, any method can be used toisolate the nucleic acid encoding the Ig or TCR variable region.

In some embodiments, the step of isolating the nucleic acid comprisesmaking a B cell or T cell hybridoma from a B cell or T cell respectivelyand isolating the nucleic acid from the hybridoma. In some embodiments,the nucleic acid is isolated using a nucleic acid amplification process.For example, in some embodiments the nucleic acid amplification processis polymerase chain reaction (PCR), ligase chain reaction (LCR), stranddisplacement amplification (SDA), transcription mediated amplification(TMA), self-sustained sequence replication (3SR), Qβ replicase basedamplification, nucleic acid sequence-based amplification (NASBA), repairchain reaction (RCR), boomerang DNA amplification (BDA) or rollingcircle amplification (RCA).

In some embodiments, the nucleic acid is isolated by sequencing therearranged Ig or TCR variable region gene in a B cell, T cell, B cellhybridoma or T cell hybridoma and synthesizing a nucleic acid sequencecomprising the rearranged Ig or TCR variable region gene. Exemplarynucleic acid sequencing processes include, but are not limited to chaintermination sequencing, sequencing by ligation, sequencing by synthesis,pyrosequencing, ion semiconductor sequencing, single-molecule real-timesequencing, 454 sequencing, and/or Dilute-'N′-Go sequencing.

When DNA fragments encoding heavy and/or light chain Ig variable regionsare obtained, these DNA fragments can be further manipulated by standardrecombinant DNA techniques, for example to convert the variable regiongenes to full-length antibody chain genes, to Fab fragment genes or to ascFv gene. In these manipulations, a variable region-encoding DNAfragment is operably linked to another DNA fragment encoding anotherprotein, such as an antibody constant region or a flexible linker. Theterm “operably linked”, as used in this context, is intended to meanthat the two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the heavy chain variable region can beconverted to a full-length heavy chain gene by operably linking thevariable region-encoding DNA to another DNA molecule encoding heavychain constant domain (CH1, CH2 and CH3). The sequences of human heavychain constant region genes are known in the art (see e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242, or Lefranc, The Immunoglobulin Handbook, London: AcademicPress 2001) and DNA fragments encompassing these regions can be obtainedby standard PCR amplification. The heavy chain constant domain can be,for example, an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constantdomain. For a Fab fragment heavy chain gene, the VH-encoding DNA can beoperably linked to another DNA molecule encoding only the heavy chainCH1 constant region.

Thus, in some embodiments, the methods described herein include the stepof operably linking a nucleic acid sequence encoding a heavy chain Igvariable domain with a nucleic acid sequence encoding a heavy chain Igconstant domain in a host cell such that the host cell expresses an Igheavy chain polypeptide comprising the Ig heavy chain variable domainand the Ig heavy chain constant domain. In some embodiments, the methodincludes the step of operably linking a nucleic acid sequence encoding alight chain Ig variable domain with a nucleic acid sequence encoding alight chain Ig constant domain in a host cell such that the host cellexpresses an Ig light chain polypeptide comprising the Ig light chainvariable domain and the Ig heavy chain constant domain. In someembodiments, the method includes the step of operably linking a nucleicacid sequence encoding a heavy chain Ig variable domain with a nucleicacid sequence encoding a heavy chain Ig constant domain in a host celland operably linking a nucleic acid sequence encoding a light chain Igvariable domain with a nucleic acid sequence encoding a light chain Igconstant domain in the host cell such that the host cell expresses anantibody having a heavy chain comprising the heavy chain Ig variabledomain and the heavy chain Ig constant domain and a light chaincomprising the light chain Ig variable domain and the light chain Igconstant domain. Ig variable regions can be linked with Ig constantregions using standard molecular biology techniques well known in theart. In some embodiments, any host cell capable of expressing animmunoglobulin polypeptide can be used. In some embodiments the cell isa CHO cell, a HEK-293 cell, a BHK cell, a NSO cell, a SP2/0 cell or aVero cell or a retinal cell expressing a viral nucleic acid sequence(e.g., PERC.6™ cell).

In some embodiments, the nucleic acid encoding the heavy chain constantdomain encodes a constant domain that comprises a modified Fc domain(e.g., a mutation that alters the interaction between the Fc and a Fcreceptor). For example, in some embodiments, the constant domaincomprises modification to its Fc domain at position 235, 236, 237, 239,265, 267, 268, 269, 270, 298, 326, 327, 330, 332, 350, 351, 366, 392,394, 405 and/or 407 (using the EU numbering system). In someembodiments, the modification is selected from the group consisting ofL235A, G236E, G237F, S239E, S239D, D265E, D265S, S267E, S267D, S267G,H268E, H268D, E269L, D270N, D270E, S298A, K326A, K326D, A327H, A327V,A327L, A330I, A330S, 1332E, T350V, L351Y, T366L, K392M, K392L, T394W,F405A and/or Y407V (using the EU numbering system). In some embodiments,the constant domain comprises multiple modifications to its Fc domain.In some embodiments, the multiple modifications are selected from thegroup consisting of D270N/K326D, S239E/S298A/K326A/A327H,L235A/S239E/D265E/A327H, G236E/G237F/S239E, G237F/S239E/D265E,G327F/S239E/H268D, G236E/D270N/A327V/1332E, G237F/S239E/A327H,G237F/A327L/A330I, S239D/D265S/S298A/I332E, S239E/D265S/H268D/I332E,S239E/D265S/I332E, S239E/S267E/H268D, S239E/A327L/A330I,D265E/S267D/A330S, S267G/H268E/D270E, H268D/E269L/S298A/K326A/A327H,H268D//K326A/A327H. Additional Fc modifications and combinations of Fcmodifications are provided in U.S. Pat. Nos. 5,624,821, 5,648,260,6,528,624, 6,737,056, 7,122,637, 7,183,387, 7,297,775, 7,317,091,7,332,581, 7,632,497, 7,662,925, 7,695,936, 8,093,359, 8,216,805,8,218,805, 8,388,955 and 8,937,158, and U.S. Patent Publication Nos.2005/0054832, 2006/0222653, 2006/0275282, 2006/0275283, 2007/0190063,2008/0154025, 2009/0042291 2013/0108623 and 2013/0089541, each of whichis hereby incorporated by reference.

Antigen Binding Proteins

In certain aspects, provided herein are antigen binding proteins (e.g.,antibodies, TCRs, CARs and antigen binding fragments thereof) obtainableand/or obtained according to a method described herein (e.g., using anon-human animal described herein).

In certain embodiments, the antigen binding molecules provided hereinare able to specifically bind a target antigen with a dissociationconstant of no greater than 10⁻⁶, 10⁻⁷, 10⁻⁸ or 10⁻⁹ M. In someembodiments, the binding affinity of the antigen binding protein to anantigen (as expressed by K_(D)) is at least 10 fold less, at least 100fold less or at least 1000 fold less than the affinity of the antigenbinding protein for an unrelated antigen. In some embodiments, theantigen binding protein binds to a peptide/MHC complex with adissociation constant of no greater than 10⁻⁶, 10⁻⁷, 10⁻⁸ or 10⁻⁹ M. Insome embodiments, the binding affinity of the antigen binding protein toa peptide/MHC complex (as expressed by K_(D)) is at least 10 fold less,at least 100 fold less or at least 1000 fold less than the affinity ofthe antigen binding protein for the peptide for the same MHC proteinpresenting an unrelated peptide. Standard assays to evaluate the bindingability of the antigen binding proteins are known in the art, includingfor example, ELISAs, Western blots and RIAs. The binding kinetics (e.g.,binding affinity) of the antigen binding protein also can be assessed bystandard assays known in the art, such as by Biacore analysis.

In some embodiments, the antigen comprises an epitope of and/or is acancer-associated antigen. Examples of cancer-associated antigensinclude, but are not limited to, adipophilin, AIM-2, ALDH1A1,alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA,carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27,CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2,cyclin D1, Cyclin-AL dek-can fusion protein, DKK1, EFTUD2, Elongationfactor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen(“ETA”), ETV6-AML1 fusion protein, EZH2, FGFS, FLT3-ITD, FN1,G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV,gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-All,HLA-A2, HLA-DOB, hsp70-2, IDOL IGF2B3, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein,Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2,MATN, MC1R, MCSP, mdm-2, MEL Melan-A/MART-1, Meloe, Midkine, MMP-2,MMP-7, MUC1, MUCSAC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I,N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ES0-1/LAGE-2, OAL OGT, OS-9, Ppolypeptide, p53, PAP, PAXS, PBF, pml-RARalpha fusion protein,polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDXS, PSA, PSMA,PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGSS, RhoC, RNF43, RU2AS, SAGE,secernin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1,survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase,TGF-betaRll, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2,TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a.In some embodiments, the antigen is a neo-antigen.

In some embodiment, the antigen comprises an epitope of and/or is anantigen expressed by an infectious pathogen. In some embodiments, thepathogen is a virus, a bacteria, a fungus, a helminth, or a protozoa.Some nonlimiting examples of viruses include HPV, HBV, hepatitis C Virus(HCV), retroviruses such as human immunodeficiency virus (HIV-1 andHIV-2), herpes viruses such as Epstein Barr Virus (EBV), cytomegalovirus(CMV), HSV-1 and HSV-2, and influenza virus. In some embodiments, theparasite is malaria. In some embodiments, pathogen is Aspergillus,Brugia, Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria,Gonococcus, Histoplasma, Leishmania, Mycobacterium, Mycoplasma,Paramecium, Pertussis, Plasmodium, Pneumococcus, Pneumocystis,Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus,Toxoplasma and Vibriocholerae. Exemplary species include Neisseriagonorrhea Mycobacterium tuberculosis, Candida albicans, Candidatropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group BStreptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granulomainguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus.Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus,Campylobacter fetus intestinalis, Leptospira pomona, Listeriamonocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonas foetus,Toxoplasma gondii, Escherichia coli, Actinobacillus equuli, Salmonellaabortus ovis, Salmonella abortus equi, Pseudomonas aeruginosa,Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis,Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramosa,Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridiumbotulinum; or, a fungus, such as, e.g., Paracoccidioides brasiliensis;or other pathogen, e.g., Plasmodium falciparum.

In some embodiments, the antigen comprises an epitope of and/or is aprotein that is the target of an autoreactive T cell in an inflammatorydisease, skin or organ transplantation rejection, graft-versus-hostdisease (GVHD), or autoimmune diseases. Examples of autoimmune diseasesinclude, for example, glomerular nephritis, arthritis, dilatedcardiomyopathy-like disease, ulceous colitis, Sjogren syndrome, Crohndisease, systemic erythematodes, chronic rheumatoid arthritis, multiplesclerosis, psoriasis, allergic contact dermatitis, polymyosiis,pachydermλ periarteritis nodosλ rheumatic fever, vitiligo vulgaris,insulin dependent diabetes mellitus, Behcet disease, Hashimoto disease,Addison disease, dermatomyositis, myasthenia gravis, Reiter syndrome,Graves' disease, anaemia perniciosλ Goodpasture syndrome, sterilitydisease, chronic active hepatitis, pemphigus, autoimmune thrombopenicpurpurλ and autoimmune hemolytic anemiλ active chronic hepatitis,Addison's disease, anti-phospholipid syndrome, atopic allergy,autoimmune atrophic gastritis, achlorhydra autoimmune, celiac disease,Cushing's syndrome, dermatomyositis, discoid lupus, erythematosis,Goodpasture's syndrome, Hashimoto's thyroiditis, idiopathic adrenalatrophy, idiopathic thrombocytopeniλ insulin-dependent diabetes,Lambert-Eaton syndrome, lupoid hepatitis, some cases of lymphopeniλmixed connective tissue disease, pemphigoid, pemphigus vulgaris,pernicious anemλ phacogenic uveitis, polyarteritis nodosλ polyglandularautosyndromes, primary biliary cirrhosis, primary sclerosingcholangitis, Raynaud's syndrome, relapsing polychondritis, Schmidt'ssyndrome, limited scleroderma (or crest syndrome), sympatheticophthalmiλ systemic lupus erythematosis, Takayasu's arteritis, temporalarteritis, thyrotoxicosis, type b insulin resistance, ulcerative colitisand Wegener's granulomatosis. Exemplary proteins include targeted byautoreactive T cells include, for example, p205, insulin,thyroid-stimulating hormone, tyrosinase, TRP1, and myelin.

In some embodiments, the antigen binding protein is an antibody. In someembodiments, the antibodies provided herein comprise human heavy chainvariable domains. In some embodiments, the antibodies comprise humanheavy chain constant domains. In some embodiments, the antibodiesprovided herein comprise a IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgDconstant domain. The sequences of human heavy chain constant domains areknown in the art (see e.g., Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242, or Lefranc, TheImmunoglobulin Handbook, London: Academic Press 2001). In someembodiments, the antibodies provided herein lack a heavy chain constantdomain or a portion thereof.

In some embodiments, the antibodies provided herein comprise a modifiedFc domain (e.g., a mutation that alters the interaction between the Fcand a Fc receptor). For example, in some embodiments, the antibodiesprovided herein comprise modification to their Fc domain at position235, 236, 237, 239, 265, 267, 268, 269, 270, 298, 326, 327, 330, 332,350, 351, 366, 392, 394, 405 and/or 407 (using the EU numbering system).In some embodiments, the modification is selected from the groupconsisting of L235A, G236E, G237F, S239E, S239D, D265E, D265S, S267E,S267D, S267G, H268E, H268D, E269L, D270N, D270E, S298A, K326A, K326D,A327H, A327V, A327L, A330I, A330S, 1332E, T350V, L351Y, T366L, K392M,K392L, T394W, F405A and/or Y407V (using the EU numbering system). Insome embodiments, the antibodies comprise multiple modifications totheir Fc domain. In some embodiments, the multiple modifications areselected from the group consisting of D270N/K326D,S239E/S298A/K326A/A327H, L235A/S239E/D265E/A327H, G236E/G237F/S239E,G237F/S239E/D265E, G327F/S239E/H268D, G236E/D270N/A327V/1332E,G237F/S239E/A327H, G237F/A327L/A330I, S239D/D265S/S298A/I332E,S239E/D265S/H268D/I332E, S239E/D265S/I332E, S239E/S267E/H268D,S239E/A327L/A330I, D265E/S267D/A330S, S267G/H268E/D270E,H268D/E269L/S298A/K326A/A327H, H268D//K326A/A327H. Additional Fcmodifications and combinations of Fc modifications are provided in U.S.Pat. Nos. 5,624,821, 5,648,260, 6,528,624, 6,737,056, 7,122,637,7,183,387, 7,297,775, 7,317,091, 7,332,581, 7,632,497, 7,662,925,7,695,936, 8,093,359, 8,216,805, 8,218,805, 8,388,955 and 8,937,158, andU.S. Patent Publication Nos. 2005/0054832, 2006/0222653, 2006/0275282,2006/0275283, 2007/0190063, 2008/0154025, 2009/0042291 2013/0108623 and2013/0089541, each of which is hereby incorporated by reference.

In some embodiments, the antibody is a bi-specific antibody. In someembodiments, the two antigen binding domains of the bi-specific antibodyhave distinct heavy chain variable domains but have identical lightchain variable domains. In some embodiments, the Fc domains of the heavychains comprise modifications to facilitate heavy chain heterodimerformation and/or to inhibit heavy chain homodimer formation. Suchmodifications are provided, for example, in U.S. Pat. Nos. 5,731,168,5,807,706, 5,821,333, 7,642,228 and 8,679,785 and in U.S. Pat. Pub. No.2013/0195849, each of which is hereby incorporated by reference.

In some embodiments, the antibodies provided herein have human lightchain variable domains. In some embodiments, the light chain variabledomains are λ light chain variable domains. In some embodiments, thelight chain variable domains are κ light chain variable domains. In someembodiments, the antibodies have human light chain constant domains. Insome embodiments, the light chain constant domains are λ light chainconstant domains. In some embodiments, the light chain constant domainsare κ light chain constant domains. The sequences of human light chainconstant domains are known in the art (see e.g., Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242, or Lefranc, The Immunoglobulin Handbook, London: Academic Press2001).

In some embodiments, the antibodies described herein are intactantibodies. In some embodiments, the antibodies described herein areantibody fragments that retain antigen binding. In some embodiments, theantibody fragment is a Fab, Fab′, F(ab′)₂, Fv, scFv, disulfide linkedFv, Fd, single-chain antibodies, isolated CDRH3 or another antibodyfragment that retain at least a portion of the variable domain of anintact antibody.

In certain embodiments, the antigen binding protein is a CAR. In someembodiments, the CAR is membrane-bound. In some embodiments, the CAR isa soluble CAR (e.g., lacking a transmembrane or cytoplasmic domain). Insome embodiments, such CARs comprise a first CAR polypeptide comprisingan Ig heavy chain variable domain and a TCRβ constant domain and asecond CAR polypeptide comprising an Ig light chain variable domain(e.g., an Ig κ variable domain or an Ig λ variable domain) and a TCRαconstant domain. In some embodiments, the Ig heavy chain variable domainand/or the Ig light chain variable domain are human Ig variable domains.In some embodiments, the TCRβ constant domain and/or the TCRα constantdomain are non-human constant domains (e.g., rat or mouse constantdomains). In some embodiments, the TCRβ constant domain and/or the TCRαconstant domain are human constant domains.

In certain embodiments, the antigen binding protein is a TCR. In someembodiments, the TCR is membrane-bound. In some embodiments, the TCR isa soluble TCR (e.g., lacking a transmembrane or cytoplasmic domain). Insome embodiments, such TCRs comprise a first TCR polypeptide comprisinga TCRβ variable domain and a TCRβ constant domain and a second TCRpolypeptide comprising a TCRα variable domain and a TCRα constantdomain. In some embodiments, the TCRα variable domain and/or the TCRβvariable domain are human TCR variable domains. In some embodiments, theTCRβ constant domain and/or the TCRα constant domain are non-humanconstant domains (e.g., rat or mouse constant domains). In someembodiments, the TCRβ constant domain and/or the TCRα constant domainare human constant domains.

Pharmaceutical Compositions

In certain embodiments, provided herein is a composition, e.g., apharmaceutical composition, containing at least one agent describedherein (e.g., an antigen binding molecule described herein, such as anantibody, a CAR or a TCR described herein, obtained from the non-humananimal described herein) formulated together with a pharmaceuticallyacceptable carrier.

The pharmaceutical compositions provided herein may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,e.g., those targeted for buccal, sublingual, and systemic absorption,boluses, powders, granules, pastes for application to the tongue; or (2)parenteral administration, for example, by subcutaneous, intramuscular,intravenous or epidural injection as, for example, a sterile solution orsuspension, or sustained-release formulation.

Pharmaceutical compositions provided herein suitable for parenteraladministration comprise one or more agents described herein incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions provided herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

In certain embodiments, the compositions comprise an antibody, a TCRand/or a CAR described herein in a concentration resulting in a w/vappropriate for a desired dose. The antibody, TCR and/or CAR may bepresent in the composition at a concentration of at least 1 mg/mL, atleast 5 mg/mL, at least 10 mg/mL, at least 15 mg/mL, at least 20 mg/mL,at least 25 mg/mL, at least 30 mg/mL, at least 35 mg/mL, at least 40mg/mL, at least 45 mg/mL, at least 50 mg/mL, at least 55 mg/mL, at least60 mg/mL, at least 65 mg/mL, at least 70 mg/mL, at least 75 mg/mL, atleast 80 mg/mL, at least 85 mg/mL, at least 90 mg/mL, at least 95 mg/mL,at least 100 mg/mL, at least 105 mg/mL, at least 110 mg/mL, at least 115mg/mL, at least 120 mg/mL, at least 125 mg/mL, at least 130 mg/mL, atleast 135 mg/mL, at least 140 mg/mL, at least 150 mg/mL, at least 200mg/mL, at least 250 mg/mL, or at least 300 mg/mL.

In some embodiments, the composition comprises one or more activecompounds as necessary for the particular indication being treated,typically those with complementary activities that do not adverselyaffect each other. Such additional active compounds are suitably presentin combination in amounts that are effective for the purpose intended.

In some embodiments, compositions are prepared by mixing an antibody, aTCR and/or a CAR described herein with optional physiologicallyacceptable carriers, excipients or stabilizers, including, but notlimited to buffering agents, saccharides, salts, surfactants,solubilizers, polyols, diluents, binders, stabilizers, salts, lipophilicsolvents, amino acids, chelators, preservatives, or the like (Goodmanand Gilman's The Pharmacological Basis of Therapeutics, 12th edition, L.Brunton, et al. and Remington's Pharmaceutical Sciences, 16th edition,Osol, A. Ed. (1999)), in the form of lyophilized compositions or aqueoussolutions at a desired final concentration. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as histidine,phosphate, citrate, glycine, acetate and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including trehalose, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g., Zn-protein complexes); and/or non-ionic surfactantssuch as TWEEN, polysorbate 80, PLURONICS® or polyethylene glycol (PEG).

In some embodiments, the buffering agent is histidine, citrate,phosphate, glycine, or acetate. The saccharide excipient may betrehalose, sucrose, mannitol, maltose or raffinose. The surfactant maybe polysorbate 20, polysorbate 40, polysorbate 80, or Pluronic F68. Thesalt may be NaCl, KCl, MgCl2 or CaCl2.

In some embodiments, the composition comprises a buffering or pHadjusting agent to provide improved pH control. Such a composition mayhave a pH of between about 3.0 and about 9.0, between about 4.0 andabout 8.0, between about 5.0 and about 8.0, between about 5.0 and about7.0, between about 5.0 and about 6.5, between about 5.5 and about 8.0,between about 5.5 and about 7.0, or between about 5.5 and about 6.5. Ina further embodiment, such a composition has a pH of about 3.0, about3.5, about 4.0, about 4.5, about 5.0, about 5.1, about 5.2, about 5.3,about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6,about 6.7, about 6.8, about 6.9, about 7.0, about 7.5, about 8.0, about8.5, or about 9.0. In a specific embodiment, a composition has a pH ofabout 6.0. One of skill in the art understands that the pH of acomposition generally should not be equal to the isoelectric point ofthe particular antibody, TCR or CAR to be used in the composition.Typically, the buffering agent is a salt prepared from an organic orinorganic acid or base. Representative buffering agents include, but arenot limited to, organic acid salts such as salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride,or phosphate buffers. In addition, amino acid components can alsofunction in a buffering capacity. Representative amino acid componentswhich may be utilized in the composition as buffering agents include,but are not limited to, glycine and histidine. In certain embodiments,the buffering agent is chosen from histidine, citrate, phosphate,glycine, and acetate. In a specific embodiment, the buffering agent ishistidine. In another specific embodiment, the buffering agent iscitrate. In yet another specific embodiment, the buffering agent isglycine. The purity of the buffering agent should be at least 98%, or atleast 99%, or at least 99.5%. As used herein, the term “purity” in thecontext of histidine and glycine refers to chemical purity of histidineor glycine as understood in the art, e.g., as described in The MerckIndex, 13th ed., O'Neil et al. ed. (Merck & Co., 2001).

In certain embodiments, the composition comprises histidine as abuffering agent. In certain embodiments the histidine is present in thecomposition at a concentration of at least about 1 mM, at least about 5mM, at least about 10 mM, at least about 20 mM, at least about 30 mM, atleast about 40 mM, at least about 50 mM, at least about 75 mM, at leastabout 100 mM, at least about 150 mM, or at least about 200 mM histidine.In another embodiment, a composition comprises between about 1 mM andabout 200 mM, between about 1 mM and about 150 mM, between about 1 mMand about 100 mM, between about 1 mM and about 75 mM, between about 10mM and about 200 mM, between about 10 mM and about 150 mM, between about10 mM and about 100 mM, between about 10 mM and about 75 mM, betweenabout 10 mM and about 50 mM, between about 10 mM and about 40 mM,between about 10 mM and about 30 mM, between about 20 mM and about 75mM, between about 20 mM and about 50 mM, between about 20 mM and about40 mM, or between about 20 mM and about 30 mM histidine. In a furtherembodiment, the composition comprises about 1 mM, about 5 mM, about 10mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM,about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In aspecific embodiment, a composition may comprise about 10 mM, about 25mM, or no histidine.

In some embodiments, the composition comprises a carbohydrate excipient.Carbohydrate excipients can act, e.g., as viscosity enhancing agents,stabilizers, bulking agents, solubilizing agents, and/or the like.Carbohydrate excipients are generally present at between about 1% toabout 99% by weight or volume, e.g., between about 0.1% to about 20%,between about 0.1% to about 15%, between about 0.1% to about 5%, betweenabout 1% to about 20%, between about 5% to about 15%, between about 8%to about 10%, between about 10% and about 15%, between about 15% andabout 20%, between 0.1% to 20%, between 5% to 15%, between 8% to 10%,between 10% and 15%, between 15% and 20%, between about 0.1% to about5%, between about 5% to about 10%, or between about 15% to about 20%. Instill other specific embodiments, the carbohydrate excipient is presentat 1%, or at 1.5%, or at 2%, or at 2.5%, or at 3%, or at 4%, or at 5%,or at 10%, or at 15%, or at 20%.

In some embodiments, the composition comprises a carbohydrate excipient.Carbohydrate excipients suitable for use in the compositions include,but are not limited to, monosaccharides such as fructose, maltose,galactose, glucose, D-mannose, sorbose, and the like; disaccharides,such as lactose, sucrose, trehalose, cellobiose, and the like;polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans,starches, and the like; and alditols, such as mannitol, xylitol,maltitol, lactitol, xylitol sorbitol (glucitol) and the like. In certainembodiments, the carbohydrate excipients for use in the compositionsprovided herein are chosen from sucrose, trehalose, lactose, mannitol,and raffinose. In a specific embodiment, the carbohydrate excipient istrehalose. In another specific embodiment, the carbohydrate excipient ismannitol. In yet another specific embodiment, the carbohydrate excipientis sucrose. In still another specific embodiment, the carbohydrateexcipient is raffinose. The purity of the carbohydrate excipient shouldbe at least 98%, or at least 99%, or at least 99.5%.

In some embodiments, the composition comprises trehalose. In certainembodiments, a composition comprises at least about 1%, at least about2%, at least about 4%, at least about 8%, at least about 20%, at leastabout 30%, or at least about 40% trehalose. In another embodiment, acomposition comprises between about 1% and about 40%, between about 1%and about 30%, between about 1% and about 20%, between about 2% andabout 40%, between about 2% and about 30%, between about 2% and about20%, between about 4% and about 40%, between about 4% and about 30%, orbetween about 4% and about 20% trehalose. In a further embodiment, acomposition comprises about 1%, about 2%, about 4%, about 6%, about 8%,about 15%, about 20%, about 30%, or about 40% trehalose. In a specificembodiment, a composition comprises about 4%, about 6% or about 15%trehalose.

In certain embodiments, the composition comprises an excipient. In aspecific embodiment, a composition comprises at least one excipientchosen from: sugar, salt, surfactant, amino acid, polyol, chelatingagent, emulsifier and preservative. In certain embodiments, acomposition comprises a salt, e.g., a salt selected from: NaCl, KCl,CaCl2, and MgCl2. In a specific embodiment, the composition comprisesNaCl.

In some embodiments, the composition comprises an amino acid, e.g.,lysine, arginine, glycine, histidine or an amino acid salt. Thecomposition may comprise at least about 1 mM, at least about 10 mM, atleast about 25 mM, at least about 50 mM, at least about 100 mM, at leastabout 150 mM, at least about 200 mM, at least about 250 mM, at leastabout 300 mM, at least about 350 mM, or at least about 400 mM of anamino acid. In another embodiment, the composition may comprise betweenabout 1 mM and about 100 mM, between about 10 mM and about 150 mM,between about 25 mM and about 250 mM, between about 25 mM and about 300mM, between about 25 mM and about 350 mM, between about 25 mM and about400 mM, between about 50 mM and about 250 mM, between about 50 mM andabout 300 mM, between about 50 mM and about 350 mM, between about 50 mMand about 400 mM, between about 100 mM and about 250 mM, between about100 mM and about 300 mM, between about 100 mM and about 400 mM, betweenabout 150 mM and about 250 mM, between about 150 mM and about 300 mM, orbetween about 150 mM and about 400 mM of an amino acid. In a furtherembodiment, a composition comprises about 1 mM, 1.6 mM, 25 mM, about 50mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300mM, about 350 mM, or about 400 mM of an amino acid.

In some embodiments, the composition comprises a surfactant. The term“surfactant” as used herein refers to organic substances havingamphipathic structures; namely, they are composed of groups of opposingsolubility tendencies, typically an oil-soluble hydrocarbon chain and awater-soluble ionic group. Surfactants can be classified, depending onthe charge of the surface-active moiety, into anionic, cationic, andnonionic surfactants. Surfactants are often used as wetting,emulsifying, solubilizing, and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.Pharmaceutically acceptable surfactants like polysorbates (e.g.,polysorbates 20 or 80); polyoxamers (e.g., poloxamer 188); Triton;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUA® series (Mona Industries, Inc.,Paterson, N. J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., PLURONICS® PF68, etc.), canoptionally be added to the compositions to reduce aggregation. Incertain embodiments, a composition comprises Polysorbate 20, Polysorbate40, Polysorbate 60, or Polysorbate 80. Surfactants are particularlyuseful if a pump or plastic container is used to administer thecomposition. The presence of a pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate. The compositionsmay comprise a polysorbate which is at a concentration ranging frombetween about 0.001% to about 1%, or about 0.001% to about 0.1%, orabout 0.01% to about 0.1%. In other specific embodiments, thecompositions comprise a polysorbate which is at a concentration of0.001%, or 0.002%, or 0.003%, or 0.004%, or 0.005%, or 0.006%, or0.007%, or 0.008%, or 0.009%, or 0.01%, or 0.015%, or 0.02%.

In some embodiments, the composition comprises other excipients and/oradditives including, but not limited to, diluents, binders, stabilizers,lipophilic solvents, preservatives, adjuvants, or the like.Pharmaceutically acceptable excipients and/or additives may be used inthe compositions provided herein. Commonly used excipients/additives,such as pharmaceutically acceptable chelators (for example, but notlimited to, EDTA, DTPA or EGTA) can optionally be added to thecompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer thecomposition.

In some embodiments, the composition comprises a preservative.Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (for example, but notlimited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl andthe like), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof can optionally beadded to the compositions at any suitable concentration such as betweenabout 0.001% to about 5%, or any range or value therein. Theconcentration of preservative used in the compositions is aconcentration sufficient to yield a microbial effect. Suchconcentrations are dependent on the preservative selected and arereadily determined by the skilled artisan.

In some embodiments, the composition is isotonic with human blood,wherein the compositions have essentially the same osmotic pressure ashuman blood. Such isotonic compositions will generally have an osmoticpressure from about 250 mOSm to about 350 mOSm. Isotonicity can bemeasured by, for example, using a vapor pressure or ice-freezing typeosmometer. Tonicity of a composition is adjusted by the use of tonicitymodifiers. “Tonicity modifiers” are those pharmaceutically acceptableinert substances that can be added to the composition to provide anisotonity of the composition. Tonicity modifiers suitable for thecompositions provided herein include, but are not limited to,saccharides, salts and amino acids.

In certain embodiments, the composition is a pyrogen-free compositionwhich is substantially free of endotoxins and/or related pyrogenicsubstances. Endotoxins include toxins that are confined inside amicroorganism and are released only when the microorganisms are brokendown or die. Pyrogenic substances also include fever-inducing,thermostable substances from the outer membrane of bacteria and othermicroorganisms. Both of these substances can cause fever, hypotensionand shock if administered to humans. Due to the potential harmfuleffects, even low amounts of endotoxins must be removed fromintravenously administered pharmaceutical drug solutions. The Food &Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units(EU) per dose per kilogram body weight in a single one-hour period forintravenous drug applications (The United States PharmacopeialConvention, Pharmacopeial Forum 26 (1):223 (2000)). When therapeuticproteins are administered in amounts of several hundred or thousandmilligrams per kilogram body weight, as can be the case with proteins ofinterest (e.g., antibodies), even trace amounts of harmful and dangerousendotoxin must be removed. In some embodiments, the endotoxin andpyrogen levels in the composition are less than 10 EU/mg, or less than 5EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01EU/mg, or less than 0.001 EU/mg.

When used for in vivo administration, the composition described hereinshould be sterile. The composition may be sterilized by varioussterilization methods, including sterile filtration, radiation, etc. Incertain embodiments, composition is filter-sterilized with apresterilized 0.22-micron filter. Sterile compositions for injection canbe formulated according to conventional pharmaceutical practice asdescribed in “Remington: The Science & Practice of Pharmacy”, 21st ed.,Lippincott Williams & Wilkins, (2005). Compositions comprising proteinsof interest (e.g., antibodies or TCRs or CARs) such as those disclosedherein, ordinarily will be stored in lyophilized form or in solution. Itis contemplated that sterile compositions comprising proteins ofinterest (e.g., antibody, TCR or CAR) are placed into a container havinga sterile access port, for example, an intravenous solution bag or vialhaving an adapter that allows retrieval of the composition, such as astopper pierceable by a hypodermic injection needle. In certainembodiments, a composition is provided as a pre-filled syringe.

In certain embodiments, the composition is a lyophilized formulation.The term “lyophilized” or “freeze-dried” includes a state of a substancethat has been subjected to a drying procedure such as lyophilization,where at least 50% of moisture has been removed.

Regardless of the route of administration selected, agents providedherein, which may be used in a suitable hydrated form, and/or thepharmaceutical compositions of the provided herein, are formulated intopharmaceutically-acceptable dosage forms by conventional methods knownto those of skill in the art.

Therapeutic Methods

In certain aspects, provided herein are methods of treating a disease ordisorder comprising administering to a subject an antigen bindingprotein (e.g., an antibody, a TCR and/or a CAR described herein, such asa fully human antibody, TCR or CAR). In some embodiments, the antibody,a TCR and/or a CAR is an antibody, a TCR and/or a CAR obtained from orobtainable using the methods described herein (e.g., using a non-humananimal described herein).

In certain embodiments, provided herein are methods of treating cancerin a subject comprising administering to the subject a pharmaceuticalcomposition described herein (e.g., a pharmaceutic compositioncomprising an antibody described herein, such as a fully human antibody,TCR or CAR described herein, obtained from the non-human animal asdescribed herein). In some embodiments, the methods described herein canbe used to treat any cancerous or pre-cancerous tumor. Cancers that maybe treated by methods and compositions described herein include, but arenot limited to, cancer cells from the bladder, blood, bone, bone marrow,brain, breast, colon, esophagus, gastrointestine, gum, head, kidney,liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis,tongue, or uterus. Nonlimiting examples of various histological types ofcancer include: neoplasm, malignant; carcinoma; carcinoma,undifferentiated; giant and spindle cell carcinoma; small cellcarcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; and roblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia.

In certain embodiments, the antibody, TCR or CAR in the pharmaceuticalcomposition administered to the subject has binding specificity for anepitope of a cancer-associated antigen (e.g., an epitope expressed bythe cancer being treated). Examples of cancer-associated antigensinclude, but are not limited to, adipophilin, AIM-2, ALDH1A1,alpha-actinin-4, alpha-fetoprotein (“AFP”), ARTC1, B-RAF, BAGE-1, BCLX(L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA-125, CALCA,carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27,CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2,cyclin D1, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, Elongationfactor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen(“ETA”), ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1,G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV,gp100/Pme117, GPNMB, HAUS3, Hepsin, HER-2/neu, HERV-K-MEL, HLA-All,HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxylesterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1also known as CCDC110, LAGE-1, LDLR-fucosyltransferaseAS fusion protein,Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2,MATN, MC1R, MCSP, mdm-2, MEL Melan-A/MART-1, Meloe, Midkine, MMP-2,MMP-7, MUC1, MUCSAC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I,N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1, NY-ESO-1/LAGE-2, OAL OGT, OS-9, Ppolypeptide, p53, PAP, PAXS, PBF, pml-RARalpha fusion protein,polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDXS, PSA, PSMA,PTPRK, RAB38/NY-MEL-1, RAGE-1, RBAF600, RGSS, RhoC, RNF43, RU2AS, SAGE,secemin 1, SIRT2, SNRPD1, SOX10, Sp17, SPA17, SSX-2, SSX-4, STEAP1,survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase,TGF-betaRll, TPBG, TRAG-3, Triosephosphate isomerase, TRP-1/gp75, TRP-2,TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-1b/GAGED2a.In some embodiments, the antigen is a neo-antigen.

In certain embodiments, provided herein are methods of treating asubject suffering from an infection, including a viral infection, afungal infection, a bacterial infection, a helminth infection, or aprotozoan infection, comprising administering to the subject apharmaceutical composition described herein (e.g., a pharmaceuticcomposition comprising an antibody, TCR or CAR described herein obtainedfrom the non-human animals described herein). Nonlimiting examples ofviral infectious diseases include HPV, HBV, hepatitis C Virus (HCV),retroviruses such as human immunodeficiency virus (HIV-1 and HIV-2),herpes viruses such as Epstein Barr Virus (EBV), cytomegalovirus (CMV),HSV-1 and HSV-2, and influenza virus. A nonlimiting example of parasiticinfection is malaria. Nonlimiting examples of bacterial, fungal andother pathogenic diseases include Aspergillus, Brugi, Candid, Chlamydi,Coccidi, Cryptococcus, Dirofilari, Gonococcus, Histoplasm, Leishmani,Mycobacterium, Mycoplasm, Paramecium, Pertussis, Plasmodium,Pneumococcus, Pneumocystis, Rickettsi, Salmonell, Shigell,Staphylococcus, Streptococcus, Toxoplasma and Vibriocholerae. Exemplaryspecies include Neisseria gonorrhe, Mycobacterium tuberculosis, Candidaalbicans, Candida tropicalis, Trichomonas vaginalis, Haemophilusvaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilusducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum,Brucella abortus. Brucella melitensis, Brucella suis, Brucella canis,Campylobacter fetus, Campylobacter fetus intestinalis, Leptospira pomon,Listeria monocytogenes, Brucella ovis, Chlamydia psittaci, Trichomonasfoetus, Toxoplasma gondii, Escherichia coli, Actinobacillus equuli,Salmonella abortus ovis, Salmonella abortus equi, Pseudomonas aeruginos,Corynebacterium equi, Corynebacterium pyogenes, Actinobaccilus seminis,Mycoplasma bovigenitalium, Aspergillus fumigatus, Absidia ramos,Trypanosoma equiperdum, Babesia caballi, Clostridium tetani, Clostridiumbotulinum; or, a fungus, such as, e.g., Paracoccidioides brasiliensis;or other pathogen, e.g., Plasmodium falciparum.

In certain embodiments, the antibody, TCR or CAR in the pharmaceuticalcomposition administered to the subject has binding specificity for anepitope of an antigen expressed by an infectious pathogen (e.g., anepitope expressed by the infectious pathogen being treated).

In some embodiments, provided herein is a method of treating aninflammatory disease, skin or organ transplantation rejection,graft-versus-host disease (GVHD), or autoimmune diseases, comprisingadministering to a subject a pharmaceutical composition described herein(e.g., a pharmaceutic composition comprising an antibody, TCR or CARdescribed herein obtained from the non-human animals described herein).Examples of autoimmune diseases include, for example, glomerularnephritis, arthritis, dilated cardiomyopathy-like disease, ulceouscolitis, Sjogren syndrome, Crohn disease, systemic erythematodes,chronic rheumatoid arthritis, multiple sclerosis, psoriasis, allergiccontact dermatitis, polymyosiis, pachyderm, periarteritis nodos,rheumatic fever, vitiligo vulgaris, insulin dependent diabetes mellitus,Behcet disease, Hashimoto disease, Addison disease, dermatomyositis,myasthenia gravis, Reiter syndrome, Graves' disease, anaemia pernicios,Goodpasture syndrome, sterility disease, chronic active hepatitis,pemphigus, autoimmune thrombopenic purpur, and autoimmune hemolyticanemi, active chronic hepatitis, Addison's disease, anti-phospholipidsyndrome, atopic allergy, autoimmune atrophic gastritis, achlorhydraautoimmune, celiac disease, Cushing's syndrome, dermatomyositis, discoidlupus, erythematosis, Goodpasture's syndrome, Hashimoto's thyroiditis,idiopathic adrenal atrophy, idiopathic thrombocytopeni,insulin-dependent diabetes, Lambert-Eaton syndrome, lupoid hepatitis,some cases of lymphopeni, mixed connective tissue disease, pemphigoid,pemphigus vulgaris, pernicious anem, phacogenic uveitis, polyarteritisnodos, polyglandular autosyndromes, primary biliary cirrhosis, primarysclerosing cholangitis, Raynaud's syndrome, relapsing polychondritis,Schmidt's syndrome, limited scleroderma (or crest syndrome), sympatheticophthalmi, systemic lupus erythematosis, Takayasu's arteritis, temporalarteritis, thyrotoxicosis, type b insulin resistance, ulcerative colitisand Wegener's granulomatosis.

In certain embodiments, the antibody, TCR or CAR in the pharmaceuticalcomposition administered to the subject has binding specificity for thetarget of an autoreactive T cell in the disease being treated (e.g., anepitope targeted by autoreactive T cells in an autoimmune disease).Exemplary proteins include targeted by autoreactive T cells include, forexample, p205, insulin, thyroid-stimulating hormone, tyrosinase, TRP1,and myelin.

The pharmaceutical compositions described herein may be delivered by anysuitable route of administration, including orally, nasally, as by, forexample, a spray, rectally, intravaginally, parenterally,intracisternally and topically, as by powders, ointments or drops,including buccally and sublingually. In certain embodiments thepharmaceutical compositions are delivered generally (e.g., via oral orparenteral administration).

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions described herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular agent employed, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

In some embodiments, the CAR and/or TCR described here are used for Tcell based therapy. For example, in certain embodiments, T cellsexpressing a CAR and/or TCR described herein are administered to asubject to induce a T cell based immune response in the subject. Methodsuseful in T cell based therapy is described in, for example, inSchumacher Nat. Rev. Immunol. 2:512-519 (2002) and Bitton et al.,Frontiers in Bioscience 4:d386-393 (1999), each of which is incorporatedby reference herein.

In some aspects, provided herein is a method of inducing an immuneresponse (e.g., a T cell based immune response) in a subject. In someembodiments, the method includes administering to the subject a cell(e.g., a human T cell, such as a CD4 T cell or a CD8 T cell) expressinga CAR or TCR described herein.

In some embodiments, the subject is a subject in need thereof. In someembodiments, the subject is a subject with cancer or a subject infectedwith a pathogen. In such embodiments, the peptide in the peptide/MHCcomplex recognized by the CAR or TCR is a peptide of a cancer antigen ora peptide from an antigen expressed by an infectious pathogen.

In some aspects, provided herein is a method of inhibiting an immuneresponse in a subject. In some embodiments, the method includesadministering to the subject a regulatory T cell (e.g., a CD4+, CD-25+and Foxp3+ regulatory T cell or a Treg17 T cell) expressing a CAR or TCRdescribed.

In some embodiments, the subject is a subject in need thereof, e.g., asubject with an autoimmune disease. In such embodiments, the T cell is aregulatory T cell (i.e., a suppressor T cell) and the peptide in thepeptide/MHC complex recognized by the TCR or CAR is a self-antigen towhich the subject is undergoing an autoimmune response.

Nucleic Acid Molecules

Provided herein are nucleic acid molecules that encode the antibodies,TCRs or CARs described herein and/or portions of antibodies, TCRs andCARs described herein. In some embodiments, the nucleic acid encodes avariable domain of an antibody, TCR or CAR described herein. The nucleicacid molecules may be present, for example, in whole cells, in a celllysate, or in a partially purified or substantially pure form.

In certain aspects, provided herein are nucleic acids encoding anantibody, TCR and/or CAR polypeptide described herein or a portionthereof. The nucleic acids may be present, for example, in whole cells,in a cell lysate, or in a partially purified or substantially pure form.Nucleic acids described herein can be obtained using standard molecularbiology techniques. For example, nucleic acid molecules described hereincan be cloned using standard PCR techniques or chemically synthesized.For nucleic acids encoding CARs, TCRs or antibodies expressed byhybridomas, cDNAs encoding each chain of the antibody, TCR or CAR madeby the hybridoma can be obtained by standard PCR amplification or cDNAcloning techniques.

In certain embodiments, provided herein are vectors that contain thenucleic acid molecules described herein. As used herein, the term“vector,” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of vector isa “plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments may be ligated. Another type of vector isa viral vector, wherein additional DNA segments may be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) can be integrated intothe genome of a host cell upon introduction into the host cell, andthereby be replicated along with the host genome. Moreover, certainvectors are capable of directing the expression of genes. Such vectorsare referred to herein as “recombinant expression vectors” (or simply,“expression vectors”).

In certain embodiments, provided herein are cells that contain a nucleicacid described herein (e.g., a nucleic acid encoding an antibody, TCR orCAR described herein or a portion thereof). The cell can be, forexample, prokaryotic, eukaryotic, mammalian, avian, murine and/or human.In certain embodiments the nucleic acid described herein is operablylinked to a transcription control element such as a promoter. In someembodiments the cell transcribes the nucleic acid described herein andthereby expresses an antibody, antigen binding fragment thereof orpolypeptide described herein. The nucleic acid molecule can beintegrated into the genome of the cell or it can be extrachromasomal.

Nucleic acid molecules provided herein can be obtained using standardmolecular biology techniques. For example, nucleic acid moleculesdescribed herein can be cloned using standard PCR techniques orchemically synthesized.

For antibodies and CAR nucleic acids described herein, once DNAfragments encoding a V_(H) and V_(L) segments are obtained, these DNAfragments can be further manipulated by standard recombinant DNAtechniques, for example to convert the variable region genes tofull-length antibody chain genes, to Fab fragment genes or to a scFvgene. In these manipulations, a V_(L)- or V_(H)-encoding DNA fragment isoperably linked to another DNA fragment encoding another protein, suchas an antibody constant region or a flexible linker. The term “operablylinked”, as used in this context, is intended to mean that the two DNAfragments are joined such that the amino acid sequences encoded by thetwo DNA fragments remain in-frame.

The isolated DNA encoding the heavy chain variable region can beconverted to a full-length heavy chain gene by operably linking theheavy chain variable region DNA to another DNA molecule encoding heavychain constant regions (e.g., CH1, CH2 and CH3). The sequences of humanheavy chain constant region genes are known in the art (see e.g., Kabat,E. A., et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242, or Lefranc, The Immunoglobulin Handbook,London: Academic Press 2001) and DNA fragments encompassing theseregions can be obtained by standard PCR amplification. The heavy chainconstant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgDconstant region. For a Fab fragment heavy chain gene, the VI-I-encodingDNA can be operably linked to another DNA molecule encoding only theheavy chain CH1 constant region.

The isolated DNA encoding the light chain variable region can beconverted to a full-length light chain gene (as well as a Fab lightchain gene) by operably linking the light chain variable region encodingDNA to another DNA molecule encoding a light chain constant region. Thesequences of human light chain constant region genes are known in theart (see e.g., Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, or Lefranc, TheImmunoglobulin Handbook, London: Academic Press 2001) and DNA fragmentsencompassing these regions can be obtained by standard PCRamplification. The light chain constant region can be a kappa or lambdaconstant region.

ADDITIONAL EXEMPLARY EMBODIMENTS

In exemplary embodiment 1, provided herein is a genetically modifiednon-human animal comprising in its genome a nucleic acid sequenceencoding human Terminal Deoxynucleotidyltransferase (hTdT).

In exemplary embodiment 2, provided herein is the genetically modifiednon-human animal of embodiment 1, wherein the nucleic acid sequenceencoding hTdT is operably linked to a transcriptional control element.

In exemplary embodiment 3, provided herein is the genetically modifiednon-human animal of embodiment 2, wherein the transcriptional controlelement drives expression of the nucleic acid sequence encoding hTdT inpro-B cells and/or pre-B cells.

In exemplary embodiment 4, provided herein is the genetically modifiednon-human animal of embodiment 2, wherein the transcriptional controlelement is selected from the group consisting of a RAG1 transcriptionalcontrol element, a RAG2 transcriptional control element, animmunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element and/or animmunoglobulin λ light chain transcriptional control element.

In exemplary embodiment 5, provided herein is the genetically modifiednon-human animal of any one of embodiments 1 to 4, wherein the non-humananimal expresses hTdT in pro-B cells and/or pre-B cells.

In exemplary embodiment 6, provided herein is the genetically modifiednon-human animal of embodiment 2, wherein the transcriptional controlelement drives expression of the nucleic acid sequence encoding hTdT inCD4/CD8 double-negative (DN) thymocytes and/or CD4/CD8 double-positive(DP) thymocytes.

In exemplary embodiment 7, provided herein is the genetically modifiednon-human animal of embodiment 2, wherein the transcriptional controlelement is a RAG1 transcriptional control element, a RAG2transcriptional control element, a TCRα transcriptional control element,a TCRβ transcriptional control element, a TCRγ transcriptional controlelement and/or a TCRδ transcriptional control element.

In exemplary embodiment 8, provided herein is the genetically modifiednon-human animal of any one of embodiments 1 to 7, wherein the non-humananimal expresses hTdT in DN thymocytes and/or DP thymocytes.

In exemplary embodiment 9, provided herein is the genetically modifiednon-human animal of any one of embodiments 1 to 8, wherein the nucleicacid sequence encoding hTdT is located at an immunoglobulin κ lightchain locus, an immunoglobulin λ light chain locus, an immunoglobulinheavy chain locus, a RAG1 locus, a RAG2 locus, a TCRα chain locus, aTCRβ chain locus, a TCRγ chain locus and/or a TCRδ chain locus.

In exemplary embodiment 10, provided herein is the genetically modifiednon-human animal of any one of embodiments 1 to 9, wherein the nucleicacid sequence encoding hTdT is not operably linked to a constitutivetranscriptional control element.

In exemplary embodiment 11, provided herein is the genetically modifiednon-human animal of any one of embodiments 1 to 10, wherein the hTdT isnot constitutively expressed.

In exemplary embodiment 12, provided herein is the genetically modifiednon-human animal of any one of embodiments 1 to 11, wherein at least 10%of the V-J immunoglobulin light chain junctions in the animal comprisenon-template additions.

In exemplary embodiment 13, provided herein is the genetically modifiednon-human animal of embodiment 12, wherein at least 20% of the V-Jimmunoglobulin light chain junctions in the animal comprise non-templateadditions.

In exemplary embodiment 14, provided herein is the genetically modifiednon-human animal of embodiment 12, wherein at least 40% of the V-Jimmunoglobulin light chain junctions in the animal comprise non-templateadditions.

In exemplary embodiment 15, provided herein is a genetically modifiednon-human animal comprising in its genome: a nucleic acid sequenceencoding an exogenous Terminal Deoxynucleotidyltransferase (TdT); and animmunoglobulin variable region comprising unrearranged humanimmunoglobulin variable region gene segments operably linked to animmunoglobulin constant region gene.

In exemplary embodiment 16, provided herein is the genetically modifiednon-human animal of embodiment 15, wherein the exogenous TdT is humanTdT.

In exemplary embodiment 17, provided herein is the genetically modifiednon-human animal of embodiment 15 or 16, wherein the nucleic acidsequence encoding the exogenous TdT is operably linked to atranscriptional control element.

In exemplary embodiment 18, provided herein is the genetically modifiednon-human animal of embodiment 17, wherein the transcriptional controlelement drives expression of the nucleic acid sequence encoding theexogenous TdT in pro-B cells and/or pre-B cells.

In exemplary embodiment 19, provided herein is the genetically modifiednon-human animal of embodiment 17, wherein the transcriptional controlelement is selected from the group consisting of a RAG1 transcriptionalcontrol element, a RAG2 transcriptional control element, animmunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element and/or animmunoglobulin λ light chain transcriptional control element.

In exemplary embodiment 20, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 19, wherein thenon-human animal expresses the exogenous TdT in pro-B cells and/or pre-Bcells.

In exemplary embodiment 21, provided herein is the genetically modifiednon-human animal of embodiment 17, wherein the transcriptional controlelement drives expression of the nucleic acid sequence encoding theexogenous TdT in CD4/CD8 double-negative (DN) thymocytes and/or CD4/CD8double-positive (DP) thymocytes.

In exemplary embodiment 22, provided herein is the genetically modifiednon-human animal of embodiment 17, wherein the transcriptional controlelement is a RAG1 transcriptional control element or a RAG2transcriptional control element.

In exemplary embodiment 23, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 22, wherein thenon-human animal expresses the exogenous TdT in DN thymocytes and/or DPthymocytes.

In exemplary embodiment 24, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 23, wherein the nucleicacid sequence encoding the exogenous TdT is located at an immunoglobulinκ light chain locus, an immunoglobulin λ light chain locus, animmunoglobulin heavy chain locus, a RAG1 locus, a RAG2 locus, a TCRαchain locus, a TCRβ chain locus, a TCRγ chain locus and/or a TCRδ chainlocus.

In exemplary embodiment 25, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 24, wherein the nucleicacid sequence encoding the exogenous TdT is not operably linked to aconstitutive transcriptional control element.

In exemplary embodiment 26, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 25, wherein theexogenous TdT is not constitutively expressed.

In exemplary embodiment 27, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 26, wherein at least10% of the V-J immunoglobulin light chain junctions in the animalcomprise non-template additions.

In exemplary embodiment 28, provided herein is the genetically modifiednon-human animal of embodiment 27, wherein at least 20% of the V-Jimmunoglobulin light chain junctions in the animal comprise non-templateadditions.

In exemplary embodiment 29, provided herein is the genetically modifiednon-human animal of embodiment 27, wherein at least 40% of the V-Jimmunoglobulin light chain junctions in the animal comprise non-templateadditions.

In exemplary embodiment 30, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 26, wherein the humanimmunoglobulin variable region gene segments are human heavy chainvariable region gene segments.

In exemplary embodiment 31, provided herein is the genetically modifiednon-human animal of embodiment 30, wherein the constant region gene is aheavy chain constant region gene.

In exemplary embodiment 32, provided herein is the genetically modifiednon-human animal of embodiment 31, wherein the heavy chain constantregion gene is a Cμ gene, a Cδ gene, a Cγ gene, a Cϵ gene or a Cα gene.

In exemplary embodiment 33, provided herein is the genetically modifiednon-human animal of embodiment 31 or 32, wherein the heavy chainconstant region gene is of endogenous species origin.

In exemplary embodiment 34, provided herein is the genetically modifiednon-human animal of embodiment 31 or 32, wherein the heavy chainconstant region gene is a mouse constant region gene.

In exemplary embodiment 35, provided herein is the genetically modifiednon-human animal of embodiment 31 or 32, wherein the heavy chainconstant region gene is a rat constant region gene.

In exemplary embodiment 36, provided herein is the genetically modifiednon-human animal of embodiment 31 or 32, wherein the heavy chainconstant region gene is a human constant region gene.

In exemplary embodiment 37, provided herein is the genetically modifiednon-human animal of embodiment 31 or 32, wherein the heavy chainconstant region gene has a human CH1 domain and non-human CH2 and CH3domains.

In exemplary embodiment 38, provided herein is the genetically modifiednon-human animal of embodiment 37, wherein the non-human CH2 and CH3domains are of endogenous species origin.

In exemplary embodiment 39, provided herein is the genetically modifiednon-human animal of embodiment 37, wherein the non-human CH2 and CH3domains are mouse CH2 and CH3 domains.

In exemplary embodiment 40, provided herein is the genetically modifiednon-human animal of embodiment 37, wherein the non-human CH2 and CH3domains are rat CH2 and CH3 domains.

In exemplary embodiment 10, provided herein is the genetically modifiednon-human animal of embodiments 15-40, wherein the animal lacks afunctional CH1 domain in an immunoglobulin heavy chain constant regionselected from IgG, IgA, IgE, IgD, or a combination thereof.

In exemplary embodiment 20, provided herein is the genetically modifiednon-human animal of any one of embodiments 31 to 41, wherein theimmunoglobulin variable region and the immunoglobulin constant regiongene are located at an endogenous immunoglobulin heavy chain locus.

In exemplary embodiment 43, provided herein is the genetically modifiednon-human animal of any one of embodiments 30 to 42, further comprisingin its genome an immunoglobulin variable region comprising unrearrangedhuman light chain variable region gene segments operably linked to asecond immunoglobulin constant region gene.

In exemplary embodiment 44, provided herein is the genetically modifiednon-human animal of embodiment 43, wherein the human immunoglobulinvariable region gene segments operably linked to the secondimmunoglobulin constant region gene are human κ chain variable regiongene segments.

In exemplary embodiment 45, provided herein is the genetically modifiednon-human animal of embodiment 43, wherein the human immunoglobulinvariable region gene segments operably linked to the secondimmunoglobulin constant region gene are human)\, chain variable regiongene segments.

In exemplary embodiment 46, provided herein is the genetically modifiednon-human animal of any one of embodiments 43 to 45, wherein the secondconstant region gene is a light chain constant region gene.

In exemplary embodiment 47, provided herein is the genetically modifiednon-human animal of embodiment 46, wherein the second constant regiongene is a κ constant region gene.

In exemplary embodiment 48, provided herein is the genetically modifiednon-human animal of embodiment 46, wherein the second constant regiongene is λ constant region gene.

In exemplary embodiment 49, provided herein is the genetically modifiednon-human animal of any one of embodiments 43 to 48, wherein the secondconstant region gene is of endogenous species origin.

In exemplary embodiment 50, provided herein is the genetically modifiednon-human animal of any one of embodiments 43 to 48, wherein the secondconstant region gene is a mouse constant region gene.

In exemplary embodiment 51, provided herein is the genetically modifiednon-human animal of any one of embodiments 43 to 48, wherein the secondconstant region gene is a rat constant region gene.

In exemplary embodiment 52, provided herein is the genetically modifiednon-human animal of any one of embodiments 43 to 48, wherein the secondconstant region gene is a human constant region gene.

In exemplary embodiment 53, provided herein is the genetically modifiednon-human animal of any one of embodiments 43 to 52, wherein theimmunoglobulin variable region operably linked to the secondimmunoglobulin constant region gene is located at an endogenousimmunoglobulin light chain locus.

In exemplary embodiment 54, provided herein is the genetically modifiednon-human animal of embodiment 53, wherein the second constant regiongene is a κ constant region gene and the endogenous immunoglobulin lightchain locus is an immunoglobulin locus.

In exemplary embodiment 55, provided herein is the genetically modifiednon-human animal of embodiment 53, wherein the second constant regiongene is λ constant region gene and the endogenous immunoglobulin lightchain locus is an immunoglobulin λ locus.

In exemplary embodiment 56, provided herein is the genetically modifiednon-human animal of any one of embodiments 30 to 42, further comprisingin its genome an immunoglobulin variable region comprising rearrangedhuman light chain variable region (V/J) gene segments operably linked toa second immunoglobulin constant region gene.

In exemplary embodiment 57, provided herein is the genetically modifiednon-human animal of embodiment 56, wherein the rearranged human lightchain variable region (V/J) gene segments operably linked to a secondimmunoglobulin constant region gene comprise a Vκ gene segments selectedfrom Wκ1-39 and Wκ3-20, rearranged to a Jκ gene segment.

In exemplary embodiment 58, provided herein is the genetically modifiednon-human animal of embodiment 57, wherein the animal comprises in itsgenome an immunoglobulin light chain variable region comprising aVκ1-39/Jκ5 or a Vκ3-20/Jκ1 sequence.

In exemplary embodiment 59, provided herein is the genetically modifiednon-human animal of any one of embodiments 30 to 42, further comprisingin its genome an immunoglobulin variable region comprising a limitedrepertoire of human light chain variable region (V and J) gene segmentsoperably linked to a second immunoglobulin constant region gene.

In exemplary embodiment 60, provided herein is the genetically modifiednon-human animal of embodiment 59, wherein the limited repertoire ofhuman light chain variable region (V and J) gene segments operablylinked to a second immunoglobulin constant region gene comprises two Vgene segments and at least two, preferably five, J gene segments.

In exemplary embodiment 61, provided herein is the genetically modifiednon-human animal of embodiment 60, wherein the two V gene segments areVκ1-39 and Vκ3-20 gene segments.

In exemplary embodiment 62, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 29, wherein the humanimmunoglobulin variable region gene segments are human light chainvariable region gene segments.

In exemplary embodiment 63, provided herein is the genetically modifiednon-human animal of embodiment 62, wherein the human immunoglobulinvariable region gene segments are human κ chain variable region genesegments.

In exemplary embodiment 64, provided herein is the genetically modifiednon-human animal of embodiment 63, wherein the human immunoglobulinvariable region gene segments are human)\, chain variable region genesegments.

In exemplary embodiment 65, provided herein is the genetically modifiednon-human animal of any one of embodiments 62 to 64, wherein theconstant region gene is a light chain constant region gene.

In exemplary embodiment 66, provided herein is the genetically modifiednon-human animal of embodiment 65, wherein the constant region gene is aκ constant region gene.

In exemplary embodiment 67, provided herein is the genetically modifiednon-human animal of embodiment 65, wherein the constant region gene is aλ constant region gene.

In exemplary embodiment 68, provided herein is the genetically modifiednon-human animal of any one of embodiments 62 to 64, wherein theconstant region gene is a heavy chain constant region gene.

In exemplary embodiment 69, provided herein is the genetically modifiednon-human animal of any one of embodiments 62 to 68, wherein theconstant region gene is of endogenous species origin.

In exemplary embodiment 70, provided herein is the genetically modifiednon-human animal of any one of embodiments 62 to 68, wherein theconstant region gene is a mouse constant region gene.

In exemplary embodiment 71, provided herein is the genetically modifiednon-human animal of any one of embodiments 62 to 68, wherein theconstant region gene is a rat constant region gene.

In exemplary embodiment 72, provided herein is the genetically modifiednon-human animal of any one of embodiments 62 to 68, wherein theconstant region gene is a human constant region gene.

In exemplary embodiment 73, provided herein is the genetically modifiednon-human animal of any one of embodiments 62 to 72, wherein theimmunoglobulin variable region and the immunoglobulin constant regiongene are located at an endogenous immunoglobulin light chain locus.

In exemplary embodiment 74, provided herein is the genetically modifiednon-human animal of embodiment 73, wherein the constant region gene is aκ constant region gene and the endogenous immunoglobulin light chainlocus is an immunoglobulin κ locus.

In exemplary embodiment 75, provided herein is the genetically modifiednon-human animal of embodiment 73, wherein the constant region gene is λconstant region gene and the endogenous immunoglobulin light chain locusis an immunoglobulin λ locus.

In exemplary embodiment 76, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 75, wherein theimmunoglobulin variable region comprises immunoglobulin variable regionintergenic sequences of human origin.

In exemplary embodiment 77, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 75, wherein theimmunoglobulin variable region comprises immunoglobulin variable regionintergenic sequences of endogenous species origin.

In exemplary embodiment 78, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 75, wherein theimmunoglobulin variable region comprises immunoglobulin variable regionintergenic sequences of mouse origin.

In exemplary embodiment 79, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 75, wherein theimmunoglobulin variable region comprises immunoglobulin variable regionintergenic sequences of rat origin.

In exemplary embodiment 80, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 79, further comprisingin its genome an inactivated endogenous immunoglobulin locus.

In exemplary embodiment 81, provided herein is the genetically modifiednon-human animal of embodiment 80, wherein the inactivated endogenousimmunoglobulin locus is an endogenous immunoglobulin heavy chain locus.

In exemplary embodiment 82, provided herein is the genetically modifiednon-human animal of embodiment 81, wherein the endogenous immunoglobulinheavy chain locus is inactivated by deletion of at least part of thevariable region of the endogenous heavy chain locus.

In exemplary embodiment 83, provided herein is the genetically modifiednon-human animal of embodiment 82, wherein the deletion of the at leastpart of the variable region comprises deletion of the J gene segments ofthe variable region.

In exemplary embodiment 84, provided herein is the genetically modifiednon-human animal of embodiment 81, wherein the endogenous immunoglobulinheavy chain locus is inactivated by deletion of at least part of theconstant region of the endogenous heavy chain locus.

In exemplary embodiment 85, provided herein is the genetically modifiednon-human animal of embodiment 84, wherein the deletion of the at leastpart of the constant region comprises deletion of the Cu gene of theconstant region.

In exemplary embodiment 86, provided herein is the genetically modifiednon-human animal of embodiment 80, wherein the inactivated endogenousimmunoglobulin locus is an endogenous immunoglobulin κ chain locus.

In exemplary embodiment 87, provided herein is the genetically modifiednon-human animal of embodiment 86, wherein the endogenous immunoglobulinκ chain locus is inactivated by deletion of at least part of thevariable region of the endogenous κ chain locus.

In exemplary embodiment 88, provided herein is the genetically modifiednon-human animal of embodiment 87, wherein the deletion of the at leastpart of the variable region comprises deletion of the J gene segments ofthe variable region.

In exemplary embodiment 89, provided herein is the genetically modifiednon-human animal of embodiment 86, wherein the endogenous immunoglobulinκ locus is inactivated by deletion of at least part of the constantregion of the endogenous κ chain locus.

In exemplary embodiment 90, provided herein is the genetically modifiednon-human animal of embodiment 89, wherein the deletion of the at leastpart of the constant region comprises deletion of the CI<gene of theconstant region.

In exemplary embodiment 91, provided herein is the genetically modifiednon-human animal of embodiment 80, wherein the inactivated endogenousimmunoglobulin locus is an endogenous immunoglobulin λ chain locus.

In exemplary embodiment 92, provided herein is the genetically modifiednon-human animal of embodiment 91, wherein the endogenous immunoglobulinλ chain locus is inactivated by deletion of at least part of a V-J-Ccluster of the endogenous λ chain locus.

In exemplary embodiment 93, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 92, wherein theunrearranged human immunoglobulin variable region gene segments undergorearrangement during B cell development to generate rearranged variableregion genes in the B cells of the non-human animal.

In exemplary embodiment 94, provided herein is the genetically modifiednon-human animal of embodiment 93, wherein at least 10% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 95, provided herein is the genetically modifiednon-human animal of embodiment 93, wherein at least 20% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 96, provided herein is the genetically modifiednon-human animal of embodiment 93, wherein at least 40% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 97, provided herein is the genetically modifiednon-human animal of any one of embodiments 93 to 96, wherein the animalexpresses antibodies comprising a variable domain encoded by therearranged variable region gene and a constant domain encoded by theconstant region gene.

In exemplary embodiment 98, provided herein is the genetically modifiednon-human animal of any one of embodiments 15 to 97 further comprising afunctional ectopic mouse Adam6 gene.

In exemplary embodiment 99, provided herein is a genetically modifiednon-human animal comprising in its genome: a nucleic acid sequenceencoding an exogenous Terminal Deoxynucleotidyltransferase (TdT); and aT cell receptor (TCR) variable region comprising unrearranged human TCRvariable region gene segments operably linked to a TCR constant regiongene.

In exemplary embodiment 100, provided herein is the genetically modifiednon-human animal of embodiment 99, wherein the exogenous TdT is humanTdT.

In exemplary embodiment 101, provided herein is the genetically modifiednon-human animal of embodiment 99 or 100, wherein the nucleic acidsequence encoding the exogenous TdT is operably linked to atranscriptional control element.

In exemplary embodiment 102, provided herein is the genetically modifiednon-human animal of embodiment 101, wherein the transcriptional controlelement drives expression of the nucleic acid sequence encoding theexogenous TdT in CD4/CD8 double-negative (DN) thymocytes and/or CD4/CD8double-positive (DP) thymocytes.

In exemplary embodiment 103, provided herein is the genetically modifiednon-human animal of embodiment 101, wherein the transcriptional controlelement is a RAG1 transcriptional control element, a RAG2transcriptional control element, a TCRα transcriptional control element,a TCRβ transcriptional control element, a TCRγ transcriptional controlelement and/or a TCRδ transcriptional control element.

In exemplary embodiment 104, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 103, wherein thenon-human animal expresses the exogenous TdT in DN thymocytes and/or DPthymocytes.

In exemplary embodiment 105, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 104, wherein thenucleic acid sequence encoding the exogenous TdT is located at a RAG1locus, a RAG2 locus, a TCRα chain locus, a TCRβ chain locus, a TCRγchain locus and/or a TCRδ chain locus.

In exemplary embodiment 106, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 105, wherein thenucleic acid sequence encoding the exogenous TdT is not operably linkedto a constitutive transcriptional control element.

In exemplary embodiment 107, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 106, wherein theexogenous TdT is not constitutively expressed.

In exemplary embodiment 108, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 107, wherein the humanTCR variable region gene segments are human TCRα variable region genesegments.

In exemplary embodiment 109, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 107, wherein the humanTCR variable region gene segments are human TCRβ variable region genesegments.

In exemplary embodiment 110, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 108, wherein the TCRconstant region gene is a TCRα constant region gene.

In exemplary embodiment 111, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 107 and 109, whereinthe TCR constant region gene is a TCRβ constant region gene.

In exemplary embodiment 112, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 111, wherein the TCRconstant region gene is of endogenous species origin.

In exemplary embodiment 113, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 111, wherein the TCRconstant region gene is a mouse constant region gene.

In exemplary embodiment 114, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 111, wherein the TCRconstant region gene is a rat constant region gene.

In exemplary embodiment 115, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 111, wherein the TCRconstant region gene is a human constant region gene.

In exemplary embodiment 116, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 115, wherein the TCRvariable region and the TCR constant region gene are located at anendogenous TCR locus.

In exemplary embodiment 117, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 108, 110, 112-115,wherein the endogenous TCR locus is an endogenous TCRα locus.

In exemplary embodiment 118, provided herein is the genetically modifiednon-human animal of any one of embodiments 99-107, 109, 111-115, whereinthe endogenous TCR locus is an endogenous TCRβ locus.

In exemplary embodiment 119, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 118, wherein the TCRvariable region comprises TCR variable region intergenic sequences ofhuman origin.

In exemplary embodiment 120, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 118, wherein the TCRvariable region comprises TCR variable region intergenic sequences ofendogenous species origin.

In exemplary embodiment 121, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 120, further comprisingin its genome an inactivated endogenous TCR locus.

In exemplary embodiment 122, provided herein is the genetically modifiednon-human animal of embodiment 121, wherein the inactivated endogenousTCRδ locus is a TCRα locus.

In exemplary embodiment 123, provided herein is the genetically modifiednon-human animal of embodiment 121, wherein the inactivated endogenousTCR locus is a TCRβ locus.

In exemplary embodiment 124, provided herein is the genetically modifiednon-human animal of any one of embodiments 99 to 123, wherein theunrearranged human TCR variable region gene segments undergorearrangement during T cell development to generate rearranged TCRvariable region genes in the T cells of the non-human animal.

In exemplary embodiment 125, provided herein is the genetically modifiednon-human animal of embodiment 124, wherein at least 10% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 126, provided herein is the genetically modifiednon-human animal of embodiment 124, wherein at least 20% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 127, provided herein is the genetically modifiednon-human animal of embodiment 124, wherein at least 40% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 128, provided herein is the genetically modifiednon-human animal of any one of embodiments 124 to 127, wherein theanimal expresses TCRs comprising a variable domain encoded by therearranged TCR variable region gene and a constant domain encoded by theTCR constant region gene.

In exemplary embodiment 129, provided herein is a genetically modifiednon-human animal comprising in its genome: a nucleic acid sequenceencoding an exogenous Terminal Deoxynucleotidyltransferase (TdT); and animmunoglobulin variable region comprising unrearranged humanimmunoglobulin variable region gene segments operably linked to a TCRconstant region gene.

In exemplary embodiment 130, provided herein is the genetically modifiednon-human animal of embodiment 129, wherein the exogenous TdT is humanTdT.

In exemplary embodiment 131, provided herein is the genetically modifiednon-human animal of embodiment 129 or 130, wherein the nucleic acidsequence encoding the exogenous TdT is operably linked to atranscriptional control element.

In exemplary embodiment 132, provided herein is the genetically modifiednon-human animal of embodiment 131, wherein the transcriptional controlelement drives expression of the nucleic acid sequence encoding theexogenous TdT in CD4/CD8 double-negative (DN) thymocytes and/or CD4/CD8double-positive (DP) thymocytes.

In exemplary embodiment 133, provided herein is the genetically modifiednon-human animal of embodiment 131, wherein the transcriptional controlelement is a RAG1 transcriptional control element, a RAG2transcriptional control element, a TCRα transcriptional control element,a TCRβ transcriptional control element, a TCRγ transcriptional controlelement and/or a TCRδ transcriptional control element.

In exemplary embodiment 134, provided herein is the genetically modifiednon-human animal of any one of embodiments 131 to 132, wherein thenon-human animal expresses the exogenous TdT in DN thymocytes and/or DPthymocytes.

In exemplary embodiment 135, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 134, wherein thenucleic acid sequence encoding the exogenous TdT is located at a RAG1locus, a RAG2 locus, a TCRα chain locus, a TCRβ chain locus, a TCRγchain locus and/or a TCRδ chain locus.

In exemplary embodiment 136, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 135, wherein thenucleic acid sequence encoding the exogenous TdT is not operably linkedto a constitutive transcriptional control element.

In exemplary embodiment 137, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 136, wherein theexogenous TdT is not constitutively expressed.

In exemplary embodiment 138, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 137, wherein the humanimmunoglobulin variable region gene segments are human light chainvariable region gene segments.

In exemplary embodiment 139, provided herein is the genetically modifiednon-human animal of embodiment 138, wherein the human light chainvariable region gene segments are κ gene segments.

In exemplary embodiment 140, provided herein is the genetically modifiednon-human animal of embodiment 138, wherein the human light chainvariable region gene segments are λ gene segments.

In exemplary embodiment 141, provided herein is the genetically modifiednon-human animal of any one of embodiments 138 to 140, wherein at least10% of the V-J immunoglobulin light chain junctions in the animalcomprise non-template additions.

In exemplary embodiment 142, provided herein is the genetically modifiednon-human animal of embodiment 141, wherein at least 20% of the V-Jimmunoglobulin light chain junctions in the animal comprise non-templateadditions.

In exemplary embodiment 143, provided herein is the genetically modifiednon-human animal of embodiment 141, wherein at least 40% of the V-Jimmunoglobulin light chain junctions in the animal comprise non-templateadditions.

In exemplary embodiment 144, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 137, wherein the humanimmunoglobulin variable region gene segments are human heavy chainvariable region gene segments.

In exemplary embodiment 145, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 143, wherein the TCRconstant region gene is a TCRα constant region gene.

In exemplary embodiment 146, provided herein is the genetically modifiednon-human animal of embodiments 145, wherein the immunoglobulin variableregion and the TCRα constant region gene are located at an endogenousTCRα locus.

In exemplary embodiment 147, provided herein is the genetically modifiednon-human animal of embodiment 144, wherein the TCR constant region geneis a TCRβ constant region gene.

In exemplary embodiment 148, provided herein is the genetically modifiednon-human animal of embodiment 147, wherein the immunoglobulin variableregion and the TCRβ constant region gene are located at an endogenousTCRβ locus.

In exemplary embodiment 149, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 148, wherein the TCRconstant region gene is of endogenous species origin.

In exemplary embodiment 150, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 148, wherein the TCRconstant region gene is a mouse constant region gene.

In exemplary embodiment 151, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 148, wherein the TCRconstant region gene is a rat constant region gene.

In exemplary embodiment 152, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 148, wherein the TCRconstant region gene is a human constant region gene.

In exemplary embodiment 153, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 152, wherein theimmunoglobulin variable region comprises immunoglobulin variable regionintergenic sequences of human origin.

In exemplary embodiment 154, provided herein is the genetically modifiednon-human animal of any one of embodiments 141 to 152, wherein theimmunoglobulin variable region comprises immunoglobulin variable regionintergenic sequences of endogenous species origin.

In exemplary embodiment 155, provided herein is the genetically modifiednon-human animal of any one of embodiments 141 to 152, wherein theimmunoglobulin variable region comprises immunoglobulin variable regionintergenic sequences of mouse origin.

In exemplary embodiment 156, provided herein is the genetically modifiednon-human animal of any one of embodiments 141 to 152, wherein theimmunoglobulin variable region comprises immunoglobulin variable regionintergenic sequences of rat origin.

In exemplary embodiment 157, provided herein is the genetically modifiednon-human animal of any one of embodiments 141 to 155, furthercomprising in its genome an inactivated endogenous immunoglobulin locus.

In exemplary embodiment 158, provided herein is the genetically modifiednon-human animal of embodiment 157, wherein the inactivated endogenousimmunoglobulin locus is an endogenous immunoglobulin heavy chain locus.

In exemplary embodiment 159, provided herein is the genetically modifiednon-human animal of embodiment 158, wherein the endogenousimmunoglobulin heavy chain locus is inactivated by deletion of at leastpart of the variable region of the endogenous heavy chain locus.

In exemplary embodiment 160, provided herein is the genetically modifiednon-human animal of embodiment 159, wherein the deletion of the at leastpart of the variable region comprises deletion of the J gene segments ofthe variable region.

In exemplary embodiment 161, provided herein is the genetically modifiednon-human animal of embodiment 158, wherein the endogenousimmunoglobulin heavy chain locus is inactivated by deletion of at leastpart of the constant region of the endogenous heavy chain locus.

In exemplary embodiment 162, provided herein is the genetically modifiednon-human animal of embodiment 161, wherein the deletion of the at leastpart of the constant region comprises deletion of the Cu gene of theconstant region.

In exemplary embodiment 163, provided herein is the genetically modifiednon-human animal of embodiment 157, wherein the inactivated endogenousimmunoglobulin locus is an endogenous immunoglobulin κ chain locus.

In exemplary embodiment 164, provided herein is the genetically modifiednon-human animal of embodiment 163, wherein the endogenousimmunoglobulin κ chain locus is inactivated by deletion of at least partof the variable region of the endogenous κ chain locus.

In exemplary embodiment 165, provided herein is the genetically modifiednon-human animal of embodiment 164, wherein the deletion of the at leastpart of the variable region comprises deletion of the J gene segments ofthe variable region.

In exemplary embodiment 166, provided herein is the genetically modifiednon-human animal of embodiment 163, wherein the endogenousimmunoglobulin κ locus is inactivated by deletion of at least part ofthe constant region of the endogenous κ chain locus.

In exemplary embodiment 167, provided herein is the genetically modifiednon-human animal of embodiment 164, wherein the deletion of the at leastpart of the constant region comprises deletion of the CI<gene of theconstant region.

In exemplary embodiment 168, provided herein is the genetically modifiednon-human animal of embodiment 157, wherein the inactivated endogenousimmunoglobulin locus is an endogenous immunoglobulin λ chain locus.

In exemplary embodiment 169, provided herein is the genetically modifiednon-human animal of embodiment 168, wherein the endogenousimmunoglobulin λ chain locus is inactivated by deletion of at least partof a V-J-C cluster of the endogenous λ chain locus.

In exemplary embodiment 170, provided herein is the genetically modifiednon-human animal of any one of embodiments 129 to 169, wherein theunrearranged human immunoglobulin variable region gene segments undergorearrangement during T cell development to generate rearrangedimmunoglobulin variable region genes in the T cells of the non-humananimal.

In exemplary embodiment 171, provided herein is the genetically modifiednon-human animal of embodiment 170, wherein at least 10% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 172, provided herein is the genetically modifiednon-human animal of embodiment 170, wherein at least 20% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 173, provided herein is the genetically modifiednon-human animal of embodiment 170, wherein at least 40% of therearranged variable region genes comprise non-template additions.

In exemplary embodiment 174, provided herein is the genetically modifiednon-human animal of any one of embodiments 170 to 173, wherein theanimal expresses chimeric antigen receptors comprising a variable domainencoded by the rearranged variable region gene and a constant domainencoded by the TCR constant region gene.

In exemplary embodiment 175, provided herein is the genetically modifiednon-human animal of any one of embodiments 1 to 174, wherein thenon-human animal is a mammal.

In exemplary embodiment 176, provided herein is the genetically modifiednon-human animal of embodiment 175, wherein the mammal is a rodent.

In exemplary embodiment 177, provided herein is the genetically modifiednon-human animal of embodiment 176, wherein the rodent is a rat or amouse.

In exemplary embodiment 178, provided herein is the genetically modifiednon-human animal of embodiment 176, wherein the rodent is a mouse.

In exemplary embodiment 179, provided herein is the genetically modifiednon-human animal of embodiment 176, wherein the rodent is a rat.

In exemplary embodiment 180, provided herein is a method inducingexpression of an antibody comprising a human variable domain, the methodcomprising exposing a genetically modified non-human animal ofembodiment 97 to an antigen such that the genetically modified non-humananimal produces an antibody comprising a human variable domain specificfor the antigen.

In exemplary embodiment 181, provided herein is a method of making a Bcell expressing an antibody comprising a human variable domain, themethod comprising: (a) exposing a genetically modified non-human animalof embodiment 97 to an antigen; and (b) obtaining a B cell expressing anantibody comprising a human variable domain specific for the antigenfrom the non-human animal.

In exemplary embodiment 182, provided herein is a method of making ahybridoma expressing an antibody comprising a human variable domain, themethod comprising: (a) exposing a genetically modified non-human animalof embodiment 97 to an antigen; (b) obtaining a B cell expressing anantibody comprising a human variable domain specific for the antigenfrom the non-human animal; and (c) making a hybridoma from the B cell ofstep (b).

In exemplary embodiment 183, provided herein is a method of making anucleic acid encoding a human immunoglobulin variable domain, the methodcomprising: (a) exposing a genetically modified non-human animal ofembodiment 97 to an antigen; and (b) obtaining a nucleic acid encoding ahuman immunoglobulin variable domain specific for the antigen from thenon-human animal.

In exemplary embodiment 184, provided herein is a method of making anucleic acid encoding a human immunoglobulin variable domain, the methodcomprising: (a) exposing a genetically modified non-human animal ofembodiment 97 to an antigen; (b) obtaining a B cell expressing anantibody comprising a human variable domain specific for the antigenfrom the non-human animal; (c) making a hybridoma from the B cell ofstep (b); and (d) obtaining a nucleic acid encoding a humanimmunoglobulin variable domain specific for the antigen from thehybridoma.

In exemplary embodiment 185, provided herein is a method of making anantibody comprising a human variable domain and a human constant domain,the method comprising: (a) exposing a genetically modified non-humananimal of embodiment 97 to an antigen; (b) obtaining a nucleic acidencoding a human immunoglobulin variable domain specific for the antigenfrom the non-human animal; (c) operably linking the nucleic acidencoding the immunoglobulin variable domain with a nucleic acid encodinga human immunoglobulin constant domain in a host cell; and (d) culturingthe host cell under conditions such that the host cell expresses a humanantibody comprising the immunoglobulin variable domain and theimmunoglobulin constant domain.

In exemplary embodiment 186, provided herein is a method for making anantibody comprising a human variable domain and a human constant domainspecific to an antigen, the method comprising: (a) exposing a non-humananimal of embodiment 97 to an antigen; (b) obtaining a B cell expressingan antibody comprising a human variable domain specific for the antigenfrom the non-human animal; (c) making a hybridoma from the B cell ofstep (b); (d) obtaining a nucleic acid encoding a human immunoglobulinvariable domain specific for the antigen from the hybridoma; (e)operably linking the nucleic acid encoding the immunoglobulin variabledomain with a nucleic acid encoding a human immunoglobulin constantdomain in a host cell; and (f) culturing the host cell under conditionssuch that the host cell expresses a human antibody comprising theimmunoglobulin variable domain and the immunoglobulin constant domain.

In exemplary embodiment 187, provided herein is a method of making a Tcell expressing a T cell receptor (TCR) comprising a human variabledomain specific to a peptide presented on a MHC, the method comprising:(a) exposing a genetically modified non-human animal of embodiment 128to an antigen comprising a peptide or a nucleic acid encoding an antigencomprising a peptide such that the peptide is presented on a MHC in thenon-human animal; and (b) obtaining a T cell expressing a TCR specificfor the peptide presented on the MHC from the genetically modifiednon-human animal of (a).

In exemplary embodiment 188, provided herein is a method of making a Tcell hybridoma expressing a T cell receptor (TCR) comprising a humanvariable domain specific to a peptide presented on a MHC, the methodcomprising: (a) exposing a genetically modified non-human animal ofembodiment 128 to an antigen comprising a peptide or a nucleic acidencoding an antigen comprising a peptide such that the peptide ispresented on a MHC in the non-human animal; (b) obtaining a T cellexpressing a TCR specific for the peptide presented on the MHC from thegenetically modified non-human animal of (a); and (c) making a T cellhybridoma from the T cell of step (b).

In exemplary embodiment 189, provided herein is a method for making anucleic acid encoding a human T cell receptor (TCR) variable domainspecific to a peptide presented on a MHC, the method comprising: (a)exposing a non-human animal of embodiment 128 to an antigen comprising apeptide or a nucleic acid encoding an antigen comprising a peptide suchthat the peptide is presented on a MHC in the non-human animal; (b)obtaining a T cell expressing a TCR specific for the peptide presentedon the MHC from the genetically modified non-human animal of (a); and(c) isolating a nucleic acid encoding a human TCR variable domain of theTCR from the T cell.

In exemplary embodiment 190, provided herein is a method for making a Tcell receptor (TCR) having a human variable domain and a human constantdomain specific to a peptide presented on a MHC, the method comprising:(a) exposing a non-human animal of embodiment 128 to an antigencomprising a peptide or a nucleic acid encoding an antigen comprising apeptide such that the peptide is presented on a MHC in the non-humananimal; (b) obtaining a T cell expressing a TCR specific for the peptidepresented on the MHC from the genetically modified non-human animal of(a); (c) isolating a nucleic acid encoding a TCR variable domain of theTCR from the T cell; and (d) operably linking the nucleic acid encodingthe TCR variable domain with a TCR constant domain in a cell such thatthe cell expresses a TCR comprising the TCR variable domain and the TCRconstant domain.

In exemplary embodiment 191, provided herein is a method of making Tcell expressing a chimeric antigen receptor (CAR) comprising a humanimmunoglobulin variable domain and an immunoglobulin constant specificto a peptide presented on a MHC, the method comprising: (a) exposing agenetically modified non-human animal of embodiment 174 to an antigencomprising a peptide or a nucleic acid encoding an antigen comprising apeptide such that the peptide is presented on a MHC in the non-humananimal; and (b) obtaining a T cell expressing a CAR specific for thepeptide presented on the MHC from the genetically modified non-humananimal of (a).

In exemplary embodiment 192, provided herein is a method of making Tcell hybridoma expressing a chimeric antigen receptor (CAR) comprising ahuman TCR variable domain and a immunoglobulin constant domain specificto a peptide presented on a MHC, the method comprising: (a) exposing agenetically modified non-human animal of embodiment 174 to an antigencomprising a peptide or a nucleic acid encoding an antigen comprising apeptide such that the peptide is presented on a MHC in the non-humananimal; (b) obtaining a T cell expressing a CAR specific for the peptidepresented on the MHC from the genetically modified non-human animal of(a); and (c) making a T cell hybridoma from the T cell of step (b).

In exemplary embodiment 193, provided herein is a method for making anucleic acid encoding a human immunoglobulin variable domain specific toa peptide presented on a MHC, the method comprising: (a) exposing anon-human animal of embodiment 174 to an antigen comprising a peptide ora nucleic acid encoding an antigen comprising a peptide such that thepeptide is presented on a MHC in the non-human animal; (b) obtaining a Tcell expressing a chimeric antigen receptor (CAR) specific for thepeptide presented on the MHC from the genetically modified non-humananimal of (a); and (c) isolating a nucleic acid encoding a human TCRvariable domain of the CAR from the T cell.

In exemplary embodiment 194, provided herein is a method for making anantibody having a human variable domain and a human constant domainspecific to a peptide presented on a MHC, the method comprising: (a)exposing a non-human animal of embodiment 174 to an antigen comprising apeptide or a nucleic acid encoding an antigen comprising a peptide suchthat the peptide is presented on a MHC in the non-human animal; (b)obtaining a T cell expressing a chimeric antigen receptor (CAR) specificfor the peptide presented on the MHC from the genetically modifiednon-human animal of (a); (c) isolating a nucleic acid encoding a humanimmunoglobulin variable domain of the CAR from the T cell; and (d)operably linking the nucleic acid encoding the human immunoglobulinvariable domain with a human immunoglobulin constant domain in a cellsuch that the cell expresses an antibody comprising the humanimmunoglobulin variable domain and the human immunoglobulin constantdomain.

In exemplary embodiment 195, provided herein is the method of any one ofembodiments 180 to 194, wherein the non-human animal is a mammal.

In exemplary embodiment 196, provided herein is the method of embodiment195, wherein the mammal is a rodent.

In exemplary embodiment 197, provided herein is the method of embodiment196, wherein the rodent is a rat or a mouse.

In exemplary embodiment 198, provided herein is an antibody generatedaccording to the method of embodiment 180, 185, 186 or 194.

In exemplary embodiment 199, provided herein is a cell generatedaccording to the method of embodiment 181, 187 or 191.

In exemplary embodiment 200, provided herein is a hybridoma generatedaccording to the method of embodiment 182, 188 or 192.

In exemplary embodiment 201, provided herein is a nucleic acid generatedaccording to the method of embodiment 183, 184, 189 or 193.

In exemplary embodiment 202, provided herein is a genetically modifiednon-human animal ES cell comprising in its genome a nucleic acidsequence encoding human Terminal Deoxynucleotidyltransferase (hTdT).

In exemplary embodiment 203, provided herein is the genetically modifiednon-human animal ES cell of embodiment 202, wherein the nucleic acidsequence encoding hTdT is operably linked to a transcriptional controlelement.

In exemplary embodiment 204, provided herein is the genetically modifiednon-human animal ES cell of embodiment 203, wherein the transcriptionalcontrol element drives expression of the nucleic acid sequence encodinghTdT in pro-B cells and/or pre-B cells.

In exemplary embodiment 205, provided herein is the genetically modifiednon-human animal ES cell of embodiment 203, wherein the transcriptionalcontrol element is selected from the group consisting of a RAG1transcriptional control element, a RAG2 transcriptional control element,an immunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element and/or animmunoglobulin λ light chain transcriptional control element.

In exemplary embodiment 206, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 202 to 204, whereinthe non-human animal expresses hTdT in pro-B cells and/or pre-B cells.

In exemplary embodiment 207, provided herein is the genetically modifiednon-human animal ES cell of embodiment 203, wherein the transcriptionalcontrol element drives expression of the nucleic acid sequence encodinghTdT in CD4/CD8 double-negative (DN) thymocytes and/or CD4/CD8double-positive (DP) thymocytes.

In exemplary embodiment 208, provided herein is the genetically modifiednon-human animal ES cell of embodiment 203, wherein the transcriptionalcontrol element is a RAG1 transcriptional control element, a RAG2transcriptional control element, a TCRα transcriptional control element,a TCRβ transcriptional control element, a TCRγ transcriptional controlelement and/or a TCR transcriptional control element.

In exemplary embodiment 209, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 202 to 207, wherein anon-human animal derived from the ES cell expresses hTdT in DNthymocytes and/or DP thymocytes.

In exemplary embodiment 210, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 202 to 209, whereinthe nucleic acid sequence encoding hTdT is located at an immunoglobulinκ light chain locus, an immunoglobulin λ light chain locus, animmunoglobulin heavy chain locus, a RAG1 locus, a RAG2 locus, a TCRαchain locus, a TCRβ chain locus, a TCRγ chain locus and/or a TCRδ chainlocus.

In exemplary embodiment 211, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 202 to 210, whereinthe nucleic acid sequence encoding hTdT is not operably linked to aconstitutive transcriptional control element.

In exemplary embodiment 212, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 202 to 211, whereinthe hTdT is not constitutively expressed in a non-human animal derivedfrom the non-human animal ES cell.

In exemplary embodiment 213, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 202 to 212, whereinat least 10% of the V-J immunoglobulin light chain junctions in anon-human animal derived from the ES cell comprise non-templateadditions.

In exemplary embodiment 214, provided herein is the genetically modifiednon-human animal ES cell of embodiment 213, wherein at least 20% of theV-J immunoglobulin light chain junctions in the animal derived from theES cell comprise non-template additions.

In exemplary embodiment 215, provided herein is the genetically modifiednon-human animal ES cell of embodiment 213, wherein at least 40% of theV-J immunoglobulin light chain junctions in the animal derived from theES cell comprise non-template additions.

In exemplary embodiment 216, provided herein is a genetically modifiednon-human animal ES cell comprising in its genome: a nucleic acidsequence encoding an exogenous Terminal Deoxynucleotidyltransferase(TdT); and an immunoglobulin variable region comprising unrearrangedhuman immunoglobulin variable region gene segments operably linked to animmunoglobulin constant region gene.

In exemplary embodiment 217, provided herein is the genetically modifiednon-human animal ES cell of embodiment 216, wherein the exogenous TdT ishuman TdT.

In exemplary embodiment 218, provided herein is the genetically modifiednon-human animal ES cell of embodiment 216 or 217, wherein the nucleicacid sequence encoding the exogenous TdT is operably linked to atranscriptional control element.

In exemplary embodiment 219, provided herein is the genetically modifiednon-human animal ES cell of embodiment 218, wherein the transcriptionalcontrol element drives expression of the nucleic acid sequence encodingthe exogenous TdT in pro-B cells and/or pre-B cells.

In exemplary embodiment 220, provided herein is the genetically modifiednon-human animal ES cell of embodiment 218, wherein the transcriptionalcontrol element is selected from the group consisting of a RAG1transcriptional control element, a RAG2 transcriptional control element,an immunoglobulin heavy chain transcriptional control element, animmunoglobulin κ light chain transcriptional control element and/or animmunoglobulin λ light chain transcriptional control element.

In exemplary embodiment 221, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 220, wherein anon-human animal derived from the ES cell expresses the exogenous TdT inpro-B cells and/or pre-B cells.

In exemplary embodiment 222, provided herein is the genetically modifiednon-human animal ES cell of embodiment 218, wherein the transcriptionalcontrol element drives expression of the nucleic acid sequence encodingthe exogenous TdT in CD4/CD8 double-negative (DN) thymocytes and/orCD4/CD8 double-positive (DP) thymocytes.

In exemplary embodiment 223, provided herein is the genetically modifiednon-human animal ES cell of embodiment 218, wherein the transcriptionalcontrol element is a RAG1 transcriptional control element or a RAG2transcriptional control element.

In exemplary embodiment 224, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 223, wherein anon-human animal derived from the ES cell expresses the exogenous TdT inDN thymocytes and/or DP thymocytes.

In exemplary embodiment 225, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 224, whereinthe nucleic acid sequence encoding the exogenous TdT is located at animmunoglobulin κ light chain locus, an immunoglobulin λ light chainlocus, an immunoglobulin heavy chain locus, a RAG1 locus, a RAG2 locus,a TCRα chain locus, a TCRβ chain locus, a TCRγ chain locus and/or a TCRδchain locus.

In exemplary embodiment 226, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 225, whereinthe nucleic acid sequence encoding the exogenous TdT is not operablylinked to a constitutive transcriptional control element.

In exemplary embodiment 227, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 226, whereinthe exogenous TdT is not constitutively expressed in a non-human animalderived from the ES cell.

In exemplary embodiment 228, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 227, whereinat least 10% of the V-J immunoglobulin light chain junctions in anon-human animal derived from the ES cell comprise non-templateadditions.

In exemplary embodiment 229, provided herein is the genetically modifiednon-human animal ES cell of embodiment 228, wherein at least 20% of theV-J immunoglobulin light chain junctions in the animal comprisenon-template additions.

In exemplary embodiment 230, provided herein is the genetically modifiednon-human animal ES cell of embodiment 228, wherein at least 40% of theV-J immunoglobulin light chain junctions in the animal comprisenon-template additions.

In exemplary embodiment 231, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 227, whereinthe human immunoglobulin variable region gene segments are human heavychain variable region gene segments.

In exemplary embodiment 232, provided herein is the genetically modifiednon-human animal ES cell of embodiment 231, wherein the constant regiongene is a heavy chain constant region gene.

In exemplary embodiment 233, provided herein is the genetically modifiednon-human animal ES cell of embodiment 232, wherein the heavy chainconstant region gene is a Cμ gene, a Cδ gene, a Cγ gene, a Cϵ gene or aCα gene.

In exemplary embodiment 234, provided herein is the genetically modifiednon-human animal ES cell of embodiment 232 or 233, wherein the heavychain constant region gene is of endogenous species origin.

In exemplary embodiment 235, provided herein is the genetically modifiednon-human animal ES cell of embodiment 232 or 233, wherein the heavychain constant region gene is a mouse constant region gene.

In exemplary embodiment 236, provided herein is the genetically modifiednon-human animal ES cell of embodiment 232 or 233, wherein the heavychain constant region gene is a rat constant region gene.

In exemplary embodiment 237, provided herein is the genetically modifiednon-human animal ES cell of embodiment 232 or 233, wherein the heavychain constant region gene is a human constant region gene.

In exemplary embodiment 238, provided herein is the genetically modifiednon-human animal ES cell of embodiment 232 or 233, wherein the heavychain constant region gene has a human CH1 domain and non-human CH2 andCH3 domains.

In exemplary embodiment 239, provided herein is the genetically modifiednon-human animal ES cell of embodiment 238, wherein the non-human CH2and CH3 domains are of endogenous species origin.

In exemplary embodiment 240, provided herein is the genetically modifiednon-human animal ES cell of embodiment 238, wherein the non-human CH2and CH3 domains are mouse CH2 and CH3 domains.

In exemplary embodiment 241, provided herein is the genetically modifiednon-human animal ES cell of embodiment 238, wherein the non-human CH2and CH3 domains are rat CH2 and CH3 domains.

In exemplary embodiment 242, provided herein is the genetically modifiednon-human animal ES cell of embodiments 216-241, wherein the animallacks a functional CH1 domain in an immunoglobulin heavy chain constantregion selected from IgG, IgA, IgE, IgD, or a combination thereof.

In exemplary embodiment 243, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 232 to 242, whereinthe immunoglobulin variable region and the immunoglobulin constantregion gene are located at an endogenous immunoglobulin heavy chainlocus.

In exemplary embodiment 244, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 231 to 243, furthercomprising in its genome an immunoglobulin variable region comprisingunrearranged human light chain variable region gene segments operablylinked to a second immunoglobulin constant region gene.

In exemplary embodiment 245, provided herein is the genetically modifiednon-human animal ES cell of embodiment 244, wherein the humanimmunoglobulin variable region gene segments operably linked to thesecond immunoglobulin constant region gene are human κ chain variableregion gene segments.

In exemplary embodiment 246, provided herein is the genetically modifiednon-human animal ES cell of embodiment 244, wherein the humanimmunoglobulin variable region gene segments operably linked to thesecond immunoglobulin constant region gene are human λ chain variableregion gene segments.

In exemplary embodiment 247, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 244 to 246, whereinthe second constant region gene is a light chain constant region gene.

In exemplary embodiment 248, provided herein is the genetically modifiednon-human animal ES cell of embodiment 247, wherein the second constantregion gene is a κ constant region gene.

In exemplary embodiment 249, provided herein is the genetically modifiednon-human animal ES cell of embodiment 247, wherein the second constantregion gene is a λ constant region gene.

In exemplary embodiment 250, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 244 to 249, whereinthe second constant region gene is of endogenous species origin.

In exemplary embodiment 251, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 244 to 249, whereinthe second constant region gene is a mouse constant region gene.

In exemplary embodiment 252, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 244 to 249, whereinthe second constant region gene is a rat constant region gene.

In exemplary embodiment 253, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 244 to 249, whereinthe second constant region gene is a human constant region gene.

In exemplary embodiment 254, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 244 to 253, whereinthe immunoglobulin variable region operably linked to the secondimmunoglobulin constant region gene and the second immunoglobulinconstant region gene are located at an endogenous immunoglobulin lightchain locus.

In exemplary embodiment 255, provided herein is the genetically modifiednon-human animal ES cell of embodiment 254, wherein the second constantregion gene is a κ constant region gene and the endogenousimmunoglobulin light chain locus is an immunoglobulin κ locus.

In exemplary embodiment 256, provided herein is the genetically modifiednon-human animal ES cell of embodiment 254, wherein the second constantregion gene is a λ constant region gene and endogenous immunoglobulinlight chain locus is an immunoglobulin λ locus.

In exemplary embodiment 257, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 231 to 243, furthercomprising in its genome an immunoglobulin variable region comprisingrearranged human light chain variable region (V/J) gene segmentsoperably linked to a second immunoglobulin constant region gene.

In exemplary embodiment 258, provided herein is the genetically modifiednon-human animal ES cell of embodiment 257, wherein the rearranged humanlight chain variable region (V/J) gene segments operably linked to asecond immunoglobulin constant region gene comprise a Vκ gene segmentsselected from Wκ1-39 and Wκ3-20, rearranged to a Jκ gene segment.

In exemplary embodiment 259, provided herein is the genetically modifiednon-human animal ES cell of embodiment 258, wherein the animal comprisesin its genome an immunoglobulin light chain variable region comprising aVκ1-39/Jκ5 or a Vκ3-20/Jκ1 sequence.

In exemplary embodiment 260, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 231 to 243, furthercomprising in its genome an immunoglobulin variable region comprising alimited repertoire of human light chain variable region (V and J) genesegments operably linked to a second immunoglobulin constant regiongene.

In exemplary embodiment 261, provided herein is the genetically modifiednon-human animal ES cell of embodiment 260, wherein the limitedrepertoire of human light chain variable region (V and J) gene segmentsoperably linked to a second immunoglobulin constant region genecomprises two V gene segments and at least two, preferably five, J genesegments.

In exemplary embodiment 262, provided herein is the genetically modifiednon-human animal ES cell of embodiment 261, wherein the two V genesegments are Vk1-39 and Vk3-20 gene segments.

In exemplary embodiment 263, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 230, whereinthe human immunoglobulin variable region gene segments are human lightchain variable region gene segments.

In exemplary embodiment 264, provided herein is the genetically modifiednon-human animal ES cell of embodiment 263, wherein the humanimmunoglobulin variable region gene segments are human κ chain variableregion gene segments.

In exemplary embodiment 265, provided herein is the genetically modifiednon-human animal ES cell of embodiment 263, wherein the humanimmunoglobulin variable region gene segments are human λ chain variableregion gene segments.

In exemplary embodiment 266, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 263 to 265, whereinthe constant region gene is a light chain constant region gene.

In exemplary embodiment 267, provided herein is the genetically modifiednon-human animal ES cell of embodiment 266, wherein the constant regiongene is a κ constant region gene.

In exemplary embodiment 268, provided herein is the genetically modifiednon-human animal ES cell of embodiment 266, wherein the constant regiongene is λ constant region gene.

In exemplary embodiment 269, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 263 to 265, whereinthe constant region gene is a heavy chain constant region gene.

In exemplary embodiment 270, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 263 to 269, whereinthe constant region gene is of endogenous species origin.

In exemplary embodiment 271, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 263 to 269, whereinthe constant region gene is a mouse constant region gene.

In exemplary embodiment 272, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 263 to 269, whereinthe constant region gene is a rat constant region gene.

In exemplary embodiment 273, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 263 to 269, whereinthe constant region gene is a human constant region gene.

In exemplary embodiment 274, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 263 to 273, whereinthe immunoglobulin variable region and the immunoglobulin constantregion gene are located at an endogenous immunoglobulin light chainlocus.

In exemplary embodiment 275, provided herein is the genetically modifiednon-human animal ES cell of embodiment 274, wherein the constant regiongene is a κ constant region gene and the endogenous immunoglobulin lightchain locus is an immunoglobulin locus.

In exemplary embodiment 276, provided herein is the genetically modifiednon-human animal ES cell of embodiment 274, wherein the constant regiongene is λ constant region gene and the endogenous immunoglobulin lightchain locus is an immunoglobulin λ locus.

In exemplary embodiment 277, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 276, whereinthe immunoglobulin variable region comprises immunoglobulin variableregion intergenic sequences of human origin.

In exemplary embodiment 278, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 276, whereinthe immunoglobulin variable region comprises immunoglobulin variableregion intergenic sequences of endogenous species origin.

In exemplary embodiment 279, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 276, whereinthe immunoglobulin variable region comprises immunoglobulin variableregion intergenic sequences of mouse origin.

In exemplary embodiment 280, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 276, whereinthe immunoglobulin variable region comprises immunoglobulin variableregion intergenic sequences of rat origin.

In exemplary embodiment 281, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 280, furthercomprising in its genome an inactivated endogenous immunoglobulin locus.

In exemplary embodiment 282, provided herein is the genetically modifiednon-human animal ES cell of embodiment 281, wherein the inactivatedendogenous immunoglobulin locus is an endogenous immunoglobulin heavychain locus.

In exemplary embodiment 283, provided herein is the genetically modifiednon-human animal ES cell of embodiment 282, wherein the endogenousimmunoglobulin heavy chain locus is inactivated by deletion of at leastpart of the variable region of the endogenous heavy chain locus.

In exemplary embodiment 284, provided herein is the genetically modifiednon-human animal ES cell of embodiment 283, wherein the deletion of theat least part of the variable region comprises deletion of the J genesegments of the variable region.

In exemplary embodiment 285, provided herein is the genetically modifiednon-human animal ES cell of embodiment 282, wherein the endogenousimmunoglobulin heavy chain locus is inactivated by deletion of at leastpart of the constant region of the endogenous heavy chain locus.

In exemplary embodiment 286, provided herein is the genetically modifiednon-human animal ES cell of embodiment 285, wherein the deletion of theat least part of the constant region comprises deletion of the Cu geneof the constant region.

In exemplary embodiment 287, provided herein is the genetically modifiednon-human animal ES cell of embodiment 281, wherein the inactivatedendogenous immunoglobulin locus is an endogenous immunoglobulin κ chainlocus.

In exemplary embodiment 288, provided herein is the genetically modifiednon-human animal ES cell of embodiment 287, wherein the endogenousimmunoglobulin chain locus is inactivated by deletion of at least partof the variable region of the endogenous κ chain locus.

In exemplary embodiment 289, provided herein is the genetically modifiednon-human animal ES cell of embodiment 288, wherein the deletion of theat least part of the variable region comprises deletion of the J genesegments of the variable region.

In exemplary embodiment 290, provided herein is the genetically modifiednon-human animal ES cell of embodiment 287, wherein the endogenousimmunoglobulin locus is inactivated by deletion of at least part of theconstant region of the endogenous κ chain locus.

In exemplary embodiment 291, provided herein is the genetically modifiednon-human animal ES cell of embodiment 290, wherein the deletion of theat least part of the constant region comprises deletion of the CI<geneof the constant region.

In exemplary embodiment 292, provided herein is the genetically modifiednon-human animal ES cell of embodiment 281, wherein the inactivatedendogenous immunoglobulin locus is an endogenous immunoglobulin λ chainlocus.

In exemplary embodiment 293, provided herein is the genetically modifiednon-human animal ES cell of embodiment 292, wherein the endogenousimmunoglobulin λ chain locus is inactivated by deletion of at least partof a V-J-C cluster of the endogenous λ chain locus.

In exemplary embodiment 294, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 216 to 293, whereinthe unrearranged human immunoglobulin variable region gene segmentsundergo rearrangement during B cell development to generate rearrangedvariable region genes in the B cells of a non-human animal derived fromthe ES cell.

In exemplary embodiment 295, provided herein is the genetically modifiednon-human animal ES cell of embodiment 294, wherein at least 10% of therearranged variable region genes comprise non-template additions in thenon-human animal.

In exemplary embodiment 296, provided herein is the genetically modifiednon-human animal ES cell of embodiment 294, wherein at least 20% of therearranged variable region genes comprise non-template additions in thenon-human animal.

In exemplary embodiment 297, provided herein is the genetically modifiednon-human animal ES cell of embodiment 294, wherein at least 40% of therearranged variable region genes comprise non-template additions in thenon-human animal.

In exemplary embodiment 298, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 294 to 297, whereinthe animal derived from the ES cell expresses antibodies comprising avariable domain encoded by the rearranged variable region gene and aconstant domain encoded by the constant region gene.

In exemplary embodiment 299, provided herein is a genetically modifiednon-human animal ES cell comprising in its genome: a nucleic acidsequence encoding an exogenous Terminal Deoxynucleotidyltransferase(TdT); and an T cell receptor (TCR) variable region comprisingunrearranged human TCR variable region gene segments operably linked toa TCR constant region gene.

In exemplary embodiment 300, provided herein is the genetically modifiednon-human animal ES cell of embodiment 299, wherein the exogenous TdT ishuman TdT.

In exemplary embodiment 301, provided herein is the genetically modifiednon-human animal ES cell of embodiment 299 or 300, wherein the nucleicacid sequence encoding the exogenous TdT is operably linked to atranscriptional control element.

In exemplary embodiment 302, provided herein is the genetically modifiednon-human animal ES cell of embodiment 301, wherein the transcriptionalcontrol element drives expression of the nucleic acid sequence encodingthe exogenous TdT in CD4/CD8 double-negative (DN) thymocytes and/orCD4/CD8 double-positive (DP) thymocytes.

In exemplary embodiment 303, provided herein is the genetically modifiednon-human animal ES cell of embodiment 301, wherein the transcriptionalcontrol element is a RAG1 transcriptional control element, a RAG2transcriptional control element, a TCRα transcriptional control element,a TCRβ transcriptional control element, a TCRγ transcriptional controlelement and/or a TCRδ transcriptional control element.

In exemplary embodiment 304, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 303, wherein anon-human animal derived from the ES cell expresses the exogenous TdT inDN thymocytes and/or DP thymocytes.

In exemplary embodiment 305, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 304, whereinthe nucleic acid sequence encoding the exogenous TdT is located at aRAG1 locus, a RAG2 locus, a TCRα chain locus, a TCRβ chain locus, a TCRγchain locus and/or a TCRδ chain locus.

In exemplary embodiment 306, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 305, whereinthe nucleic acid sequence encoding the exogenous TdT is not operablylinked to a constitutive transcriptional control element.

In exemplary embodiment 307, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 306, whereinthe exogenous TdT is not constitutively expressed by a non-human animalderived from the ES cell.

In exemplary embodiment 308, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 307, whereinthe human TCR variable region gene segments are human TCRα variableregion gene segments.

In exemplary embodiment 309, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 307, whereinthe human TCR variable region gene segments are human TCRβ variableregion gene segments.

In exemplary embodiment 310, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 308, whereinthe TCR constant region gene is a TCRα constant region gene.

In exemplary embodiment 311, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 307 and 309,wherein the TCR constant region gene is a TCRβ constant region gene.

In exemplary embodiment 312, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 311, whereinthe TCR constant region gene is of endogenous species origin.

In exemplary embodiment 313, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 311, whereinthe TCR constant region gene is a mouse constant region gene.

In exemplary embodiment 314, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 311, whereinthe TCR constant region gene is a rat constant region gene.

In exemplary embodiment 315, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 311, whereinthe TCR constant region gene is a human constant region gene.

In exemplary embodiment 316, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 315, whereinthe TCR variable region and the TCR constant region gene are located atan endogenous TCR locus.

In exemplary embodiment 317, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 308, 310,313-315, wherein the endogenous TCR locus is an endogenous TCRα locus.

In exemplary embodiment 318, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299-307, 309,311-315, wherein the endogenous TCR locus is an endogenous TCRβ locus.

In exemplary embodiment 319, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 318, whereinthe TCR variable region comprises TCR variable region intergenicsequences of human origin.

In exemplary embodiment 320, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 318, whereinthe TCR variable region comprises TCR variable region intergenicsequences of endogenous species origin.

In exemplary embodiment 321, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 320, furthercomprising in its genome an inactivated endogenous TCRδ locus.

In exemplary embodiment 322, provided herein is the genetically modifiednon-human animal ES cell of embodiment 321, wherein the inactivatedendogenous TCRδ locus is a TCRα locus.

In exemplary embodiment 323, provided herein is the genetically modifiednon-human animal ES cell of embodiment 321, wherein the inactivatedendogenous TCRδ locus is a TCRβ locus.

In exemplary embodiment 324, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 299 to 323, whereinthe unrearranged human TCR variable region gene segments undergorearrangement during T cell development to generate rearranged TCRvariable region genes in the T cells of a non-human animal derived fromthe ES cell.

In exemplary embodiment 325, provided herein is the genetically modifiednon-human animal ES cell of embodiment 324, wherein at least 10% of therearranged variable region genes comprise non-template additions in anon-human animal derived from the ES cell.

In exemplary embodiment 326, provided herein is the genetically modifiednon-human animal ES cell of embodiment 324, wherein at least 20% of therearranged variable region genes comprise non-template additions in anon-human animal derived from the ES cell.

In exemplary embodiment 327, provided herein is the genetically modifiednon-human animal ES cell of embodiment 324, wherein at least 40% of therearranged variable region genes comprise non-template additions in anon-human animal derived from the ES cell.

In exemplary embodiment 328, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 324 to 327, whereinthe animal derived from the ES cell expresses TCRs comprising a variabledomain encoded by the rearranged TCR variable region gene and a constantdomain encoded by the TCR constant region gene.

In exemplary embodiment 329, provided herein is a genetically modifiednon-human animal comprising in its genome: a nucleic acid sequenceencoding an exogenous Terminal Deoxynucleotidyltransferase (TdT); and animmunoglobulin variable region comprising unrearranged humanimmunoglobulin variable region gene segments operably linked to a TCRconstant region gene.

In exemplary embodiment 330, provided herein is the genetically modifiednon-human animal ES cell of embodiment 329, wherein the exogenous TdT ishuman TdT.

In exemplary embodiment 331, provided herein is the genetically modifiednon-human animal ES cell of embodiment 329 or 330, wherein the nucleicacid sequence encoding the exogenous TdT is operably linked to atranscriptional control element.

In exemplary embodiment 332, provided herein is the genetically modifiednon-human animal ES cell of embodiment 331, wherein the transcriptionalcontrol element drives expression of the nucleic acid sequence encodingthe exogenous TdT in CD4/CD8 double-negative (DN) thymocytes and/orCD4/CD8 double-positive (DP) thymocytes.

In exemplary embodiment 333, provided herein is the genetically modifiednon-human animal ES cell of embodiment 331, wherein the transcriptionalcontrol element is a RAG1 transcriptional control element, a RAG2transcriptional control element, a TCRα transcriptional control element,a TCRβ transcriptional control element, a TCRγ transcriptional controlelement and/or a TCRδ transcriptional control element.

In exemplary embodiment 334, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 333, wherein anon-human animal derived from the ES cell expresses the exogenous TdT inDN thymocytes and/or DP thymocytes.

In exemplary embodiment 335, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 334, whereinthe nucleic acid sequence encoding the exogenous TdT is located at aRAG1 locus, a RAG2 locus, a TCRα chain locus, a TCRβ chain locus, a TCRγchain locus and/or a TCRδ chain locus.

In exemplary embodiment 336, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 335, whereinthe nucleic acid sequence encoding the exogenous TdT is not operablylinked to a constitutive transcriptional control element.

In exemplary embodiment 337, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 336, whereinthe exogenous TdT is not constitutively expressed in non-human animalsderived from the ES cell.

In exemplary embodiment 338, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 337, whereinthe human immunoglobulin variable region gene segments are human lightchain variable region gene segments.

In exemplary embodiment 339, provided herein is the genetically modifiednon-human animal ES cell of embodiment 338, wherein at least 10% of theV-J immunoglobulin light chain junctions in a non-human animal derivedfrom the ES cell comprise non-template additions.

In exemplary embodiment 340, provided herein is the genetically modifiednon-human animal ES cell of embodiment 339, wherein at least 20% of theV-J immunoglobulin light chain junctions in the animal comprisenon-template additions.

In exemplary embodiment 341, provided herein is the genetically modifiednon-human animal ES cell of embodiment 339, wherein at least 40% of theV-J immunoglobulin light chain junctions in the animal comprisenon-template additions.

In exemplary embodiment 342, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 338 to 341, whereinthe human light chain variable region gene segments are κ gene segments.

In exemplary embodiment 343, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 338 to 341, whereinthe human light chain variable region gene segments are λ gene segments.

In exemplary embodiment 344, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 337, whereinthe human immunoglobulin variable region gene segments are human heavychain variable region gene segments.

In exemplary embodiment 345, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 343, whereinthe TCR constant region gene is a TCRα constant region gene.

In exemplary embodiment 346, provided herein is the genetically modifiednon-human animal ES cell of embodiment 345, wherein the immunoglobulinvariable region and the TCRα constant region gene are located at anendogenous TCRα locus.

In exemplary embodiment 347, provided herein is the genetically modifiednon-human animal ES cell of embodiment 344, wherein the TCR constantregion gene is a TCRβ constant region gene.

In exemplary embodiment 348, provided herein is the genetically modifiednon-human animal ES cell of embodiment 347, wherein the immunoglobulinvariable region and the TCRβ constant region gene are located at anendogenous TCRβ locus.

In exemplary embodiment 349, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 344, whereinthe TCR constant region gene is of endogenous species origin.

In exemplary embodiment 350, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 348, whereinthe TCR constant region gene is a mouse constant region gene.

In exemplary embodiment 351, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 348, whereinthe TCR constant region gene is a rat constant region gene.

In exemplary embodiment 352, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 348, whereinthe TCR constant region gene is a human constant region gene.

In exemplary embodiment 353, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 352, whereinthe immunoglobulin variable region comprises immunoglobulin variableregion intergenic sequences of human origin.

In exemplary embodiment 354, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 340 to 352, whereinthe immunoglobulin variable region comprises immunoglobulin variableregion intergenic sequences of endogenous species origin.

In exemplary embodiment 355, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 341 to 352, whereinthe immunoglobulin variable region comprises immunoglobulin variableregion intergenic sequences of mouse origin.

In exemplary embodiment 356, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 341 to 352, whereinthe immunoglobulin variable region comprises immunoglobulin variableregion intergenic sequences of rat origin.

In exemplary embodiment 357, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 341 to 356, furthercomprising in its genome an inactivated endogenous immunoglobulin locus.

In exemplary embodiment 358, provided herein is the genetically modifiednon-human animal ES cell of embodiment 357, wherein the inactivatedendogenous immunoglobulin locus is an endogenous immunoglobulin heavychain locus.

In exemplary embodiment 359, provided herein is the genetically modifiednon-human animal ES cell of embodiment 358, wherein the endogenousimmunoglobulin heavy chain locus is inactivated by deletion of at leastpart of the variable region of the endogenous heavy chain locus.

In exemplary embodiment 360, provided herein is the genetically modifiednon-human animal ES cell of embodiment 359, wherein the deletion of theat least part of the variable region comprises deletion of the J genesegments of the variable region.

In exemplary embodiment 361, provided herein is the genetically modifiednon-human animal ES cell of embodiment 358, wherein the endogenousimmunoglobulin heavy chain locus is inactivated by deletion of at leastpart of the constant region of the endogenous heavy chain locus.

In exemplary embodiment 362, provided herein is the genetically modifiednon-human animal ES cell of embodiment 361, wherein the deletion of theat least part of the constant region comprises deletion of the Cu geneof the constant region.

In exemplary embodiment 363, provided herein is the genetically modifiednon-human animal ES cell of embodiment 357, wherein the inactivatedendogenous immunoglobulin locus is an endogenous immunoglobulin κ chainlocus.

In exemplary embodiment 364, provided herein is the genetically modifiednon-human animal ES cell of embodiment 363, wherein the endogenousimmunoglobulin chain locus is inactivated by deletion of at least partof the variable region of the endogenous κ chain locus.

In exemplary embodiment 365, provided herein is the genetically modifiednon-human animal ES cell of embodiment 364, wherein the deletion of theat least part of the variable region comprises deletion of the J genesegments of the variable region.

In exemplary embodiment 366, provided herein is the genetically modifiednon-human animal ES cell of embodiment 365, wherein the endogenousimmunoglobulin κ locus is inactivated by deletion of at least part ofthe constant region of the endogenous κ chain locus.

In exemplary embodiment 367, provided herein is the genetically modifiednon-human animal ES cell of embodiment 366, wherein the deletion of theat least part of the constant region comprises deletion of the CI<geneof the constant region.

In exemplary embodiment 368, provided herein is the genetically modifiednon-human animal ES cell of embodiment 357, wherein the inactivatedendogenous immunoglobulin locus is an endogenous immunoglobulin λ chainlocus.

In exemplary embodiment 369, provided herein is the genetically modifiednon-human animal ES cell of embodiment 368, wherein the endogenousimmunoglobulin λ chain locus is inactivated by deletion of at least partof a V-J-C cluster of the endogenous λ chain locus.

In exemplary embodiment 370, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 331 to 369, whereinthe unrearranged human immunoglobulin variable region gene segmentsundergo rearrangement during T cell development to generate rearrangedimmunoglobulin variable region genes in the T cells of a non-humananimal derived from the ES cell.

In exemplary embodiment 371, provided herein is the genetically modifiednon-human animal ES cell of embodiment 370, wherein at least 10% of therearranged variable region genes in the non-human animal derived fromthe ES cell comprise non-template additions.

In exemplary embodiment 372, provided herein is the genetically modifiednon-human animal ES cell of embodiment 370, wherein at least 20% of therearranged variable region genes in the non-human animal derived fromthe ES cell comprise non-template additions.

In exemplary embodiment 373, provided herein is the genetically modifiednon-human animal ES cell of embodiment 370, wherein at least 40% of therearranged variable region genes in the non-human animal derived fromthe ES cell comprise non-template additions.

In exemplary embodiment 374, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 370 to 372, whereinthe non-human animal derived from the ES cell expresses chimeric antigenreceptors comprising a variable domain encoded by the rearrangedvariable region gene and a constant domain encoded by the TCR constantregion gene.

In exemplary embodiment 375, provided herein is the genetically modifiedES cell of any one of embodiments 202 to 374, further comprising afunctional ectopic mouse Adam6 gene.

In exemplary embodiment 376, provided herein is the genetically modifiednon-human animal ES cell of any one of embodiments 202 to 375, whereinthe non-human animal is a mammal.

In exemplary embodiment 377, provided herein is the genetically modifiednon-human animal ES cell of embodiment 376, wherein the mammal is arodent.

In exemplary embodiment 378, provided herein is the genetically modifiednon-human animal ES cell of embodiment 377, wherein the rodent is a rator a mouse.

In exemplary embodiment 379, provided herein is the genetically modifiednon-human animal ES cell of embodiment 377, wherein the rodent is amouse.

In exemplary embodiment 380, provided herein is the genetically modifiednon-human animal ES cell of embodiment 377, wherein the rodent is a rat.

In exemplary embodiment 381, provided herein is a method of making agenetically modified non-human animal comprising using the geneticallymodified non-human animal ES cell of any one of embodiments 202 to 380.

In exemplary embodiment 382, provided herein is a method of making anon-human animal comprising a genetic modification comprisingengineering the non-human animal to comprise in its germline a nucleicacid sequence encoding human Terminal Deoxynucleotidyltransferase(hTdT).

In exemplary embodiment 383, provided herein is a method of making anon-human animal comprising a genetic modification comprisingengineering the non-human animal to comprise in its germline: a nucleicacid sequence encoding an exogenous Terminal Deoxynucleotidyltransferase(TdT); and an immunoglobulin variable region comprising unrearrangedhuman immunoglobulin variable region gene segments operably linked to animmunoglobulin constant region gene.

In exemplary embodiment 385, provided herein is a method of making anon-human animal comprising a genetic modification comprisingengineering the non-human animal to comprise in its germline: a nucleicacid sequence encoding an exogenous Terminal Deoxynucleotidyltransferase(TdT); and a T cell receptor (TCR) variable region comprisingunrearranged human TCR variable region gene segments operably linked toa TCR constant region gene.

In exemplary embodiment 386, provided herein is a method of making anon-human animal comprising a genetic modification comprisingengineering the non-human animal to comprise in its germline: a nucleicacid sequence encoding an exogenous Terminal Deoxynucleotidyltransferase(TdT); and an immunoglobulin variable region comprising unrearrangedhuman immunoglobulin variable region gene segments operably linked to aTCR constant region gene.

In exemplary embodiment 387, provided herein is the method of any one ofembodiments 382 to 385, wherein the non-human animal is a mammal.

In exemplary embodiment 388, provided herein is the method of embodiment386, wherein the mammal is a rodent.

In exemplary embodiment 389, provided herein is the method of embodiment387, wherein the rodent is a rat or a mouse.

In exemplary embodiment 390, provided herein is the method of embodiment387, wherein the rodent is a mouse.

EXAMPLES

The invention will be further illustrated by the following nonlimitingexamples. These Examples are set forth to aid in the understanding ofthe invention but are not intended to, and should not be construed to,limit its scope in any way. The Examples do not include detaileddescriptions of conventional methods that would be well known to thoseof ordinary skill in the art (molecular cloning techniques, etc.).Unless indicated otherwise, parts are parts by weight, molecular weightis average molecular weight, temperature is indicated in Celsius, andpressure is at or near atmospheric.

Example 1 Generation of Mice Expressing Human TdT

Mice comprising a human TdT gene, either as random transgene or targetedto the immunoglobulin kappa locus, are made using VELOCIGENE® geneticengineering technology (see, e.g., U.S. Pat. No. 6,586,251 andValenzuelλ D. M., et al. (2003) High-throughput engineering of the mousegenome coupled with high-resolution expression analysis. Nat. Biotech.21(6): 652-659), wherein human sequences derived from BAC librariesusing bacterial homologous recombination are used to make largetargeting vectors (LTVECs) comprising genomic fragments of human TdTlocus and, in the case of TdT targeted to the IgK locus, flanked bytargeting arms to target the LTVECs to the IgK locus in mouse ES cells.LTVECs are linearized and electroporated into a mouse ES cell lineaccording to Valenzuela et al. ES cells are screened by TAQMAN® todetermine either gene copy number (for randomly integrated transgenes)or correct targeting to the IgK locus.

Alternatively, a short isoform human TdT (TdTS) cDNA is synthesized denovo (Blue Heron Bio) and incorporated into a targeting for introductioninto ES cells as described above.

Targeted ES cell clones are introduced into 8-cell stage (or earlier)mouse embryos by the VELOCIMOUSE® method (Poueymirou, W. T. et al.(2007). F0 generation mice fully derived from gene-targeted embryonicstem cells allowing immediate phenotypic analyses. Nat. Biotech. 25:91-99). VELOCIMICE® (FO mice fully derived from the donor ES cell)bearing human TdT locus are identified by screening by TAQMAN® in a gainof human allele assay (Valenzuela et al.). F0 pups are genotyped andbred to homozygosity. Mice homozygous for human TdT locus are made andphenotyped. As specifically described in Example 1 below, micecomprising human TdTS and unrearranged human variable light and heavychain loci are generated by introducing human TdTS random transgene ortargeting human TdTS to IgK locus in VELOCIMMUNEO mice comprising afunctional ectopic mouse Adam6 gene. However, at least for the randomlyintroduced human TdTS transgene, such animals can also be generated byfirst generating ES cells comprising a human TdTS as described below,generating mice therefrom, and breeding mice comprising randomlyintegrated human TdTS locus with VELOCIMMUNE® mice comprising afunctional ectopic mouse Adam6 gene.

Example 1.1 Generation of Transgene Expressing Short Isoform of HumanTdT (TdTS) Under Control of Mouse Rag Regulatory Elements (Rag-TdT tg)

Briefly, a large targeting vector (LTVEC), shown in detail in FIG. 1,was constructed from mouse and human BAC clones in which the mouse Rag2gene (from the ATG start codon in exon 3 to the TGA stop codon in exon 3was replaced with the human Terminal deoxynucleotidyl Transferase (TdTor DNTT) gene encoding only the short isoform, TdTS (from the ATG startcodon in exon 1 to ˜0.5 kb 3′ of the polyA signal in exon 13). The RNAsplice sites of TdT exons 7 and 12 were mutated to prevent expression ofthe long isoforms (TdTL1 and L2). In the same LTVEC, the mouse Rag1 genefrom the ATG start codon in exon 2 to the TAA stop codon in exon 2 wasreplaced with the coding sequence of the enhanced green fluorescentprotein (EGFP) and the LacZ 3′UTR-polyA signal. The LTVEC contains 130kb of upstream regulatory sequences 5′ of the mouse Rag2 gene and 8.8 kbof upstream regulatory sequences 3′ of the mouse Rag1 gene, as well asthe 5.6 kb Rag2-Rag1 intergenic region.

The LTVEC was constructed from mouse BAC clone RP23-374f10(Invitrogen/Life Technologies) containing the Rag2 and Rag1 genes, andhuman BAC clone RP11-1070o2 (Invitrogen/Life Technologies) containingthe TdT gene using standard molecular biology and recombineeringtechniques such as PCR, restriction digestion/ligation, GibsonIsothermal Assembly, CRISPR/Cas9, bacterial homologous recombination,etc. The final LTVEC contains, from 5′ to 3′: (1) aLoxp-pgkp-em7-neo-loxp cassette for selection in ES cells or bacteria,(2) 134,069 bp of mouse genomic sequence beginning 129,440 bp upstreamof Rag2 exon 1 and ending 25 bp from the start of Rag2 exon 3 (mousegenome coordinates 2:101,495,278-101,629,347 based on GRCm38 assembly),(3) 34,573 bp of human TdT genomic sequence beginning at the ATG startcodon in exon 1 and ending 514 bp 3′ of the polyA signal (human genomecoordinates 10:96,304,498-96,339,063 based on GRCh38 assembly); withinthe human TdT gene, the splice donor site of exon 7 is deleted andreplaced by a NotI site to prevent expression of the TdTL2 isoform, andthe splice acceptor site of exon 12 is deleted to prevent expression ofthe TdTL1 isoform (detailed description below), (4) AsiSI site; (5)10,742 bp of mouse genomic sequence (GRCm38 genome coordinates2:101,630,931-101,641,672) containing the 3′UTR of Rag2 exon 3 (1599bp), the 5753 bp Rag2/Rag1 intergenic region, and the 3′UTR of Rag1 exon2 (3390 bp), (6) FseI site, (7) 1,068 bp on the minus strand containingthe 249 bp polyA signal of LacZ and the 793 bp CDS of EGFP, (8) 13,459bp of mouse genomic sequence (GRCm38 coordinates2:101,644,793-101,658,251) with Rag1 on the minus strand, beginning atthe ATG start codon in Rag1 exon 2 and ending 8,750 bp 3′ of thetranscription start site of Rag1, and (9) Em7-CM cassette for selectionin bacteria (see FIG. 1).

In detail, the cloning steps to create two modifications of the TdT geneto prevent alternative splicing using TdT exons 7 and 12 (which are usedto make the long isoforms TdTL2 and TdTL1, respectively) while stillallowing splicing of the transcript encoding the short isoform TdTS wereconstructed from BAC clone RP11-1070o2 using BHR and ligation:

-   (1) 13 bp including the splice donor site of exon 7 (GTCGGGTCGTGGT    (SEQ ID NO:1), splice donor underlined) were deleted and replaced by    a NotI site (GCGGCCGC (SEQ ID NO:2)). This created an overlapping    SacII site (CCGCGG (SEQ ID NO:3)), and-   (2) The 2 bp splice acceptor site of exon 12 was deleted.

The final LTVEC is depicted in FIG. 1, with the approximate positions ofvarious sequence junctions indicated in the figure. The junctions arealso summarized in Table 1 below.

TABLE 1 Sequence Junctions of Rag-TdT Tg LTVEC SEQ ID Junction NOSequence 1 (mouse 4 TATTGCGTTTTTTTAATCCTTTCAGATAAAAGACCTA Rag2/ TTCACAATCAAAA/ATGGATCCACCACGAGCGTCCC human ACTTGAGCCCTCGGAAGAAGAGACCCCTdT) 2 (human 5 GCCCTGGCTGAGGGAAATTTTGGAACTCCCAGGC TdT/TCCAGACCCATTCTTT/GCGATCGC/TTTAGCAAA AsiS1/AGCCCCTCAGACTCAGGTATATTGCTCTCT mouse GAATCTACTTT Rag2) 3 (mouse 6CCAAAGGAAAACACATTGGCAAATACCAACTTCTATG Rag1/Fse  TGGAGATCCTAT/GGCCGGCC/1/EGFP) GGGGATCCAGACATGA TAAGATACATTGATGAGTTTGGACAAACCACAAC 4 (EGFP/ 7TCGACCAGGATGGGCACCACCCCGGTGAACAGCTC mouse CTCGCCCTTGCTCAC/CATGTTGGCTAAGCRag1) TACCTGGGAACAATGGGGGGGGGGGGGGGA GTCAAG

The final LTVEC was linearized and electroporated into VELOCIMMUNE® EScells that comprise a functional ectopic mouse Adam6 gene (see, e.g.,U.S. Pat. No. 8,642,835, incorporated herein by reference). Afterselection with Neo, ES cells were screened by TAQMAN® to determine copynumber of the transgene. ES cells comprising a single copy, two copies,or multiple copies of the human Rag-TdT transgene were obtained.

The integration site of the Rag-TdT transgene is determined via methodsknown in the art. In one embodiment, the integration site is determinedusing low coverage paired-end sequencing of the whole mouse genome(Sequencing library—Nextera DNA Library Preparation, Illumina;Sequence—Miseq, Illumina). For example, in one instance it wasdetermined that Rag-TdT transgene integrated as two tandem head to tailcopies between coordinates 41130492 and 41130502 on chromosome 1(coordinates in GRCm38/mm10 Assembly), without disrupting any codingregions.

Example 1.2 Generation of Targeted Immunoglobulin Kappa Locus Insertionof Short Isoform of Human TdT (TdTS) Under Control of Mouse RagRegulatory Elements (Rag-TdT IgK)

In order to generate a mouse comprising human TdT under control of theRag regulatory elements on the mouse immunoglobulin kappa locus, mouseIgK homology arms for recombination in ES cells were added to the 5′ and3′ ends of the construct that is generated as described in Example 1.1and FIG. 1. In general, the mouse IgK arms are added by: (1) modifyingthe mouse IgK BAC to by bacterial homologous recombination to insert aselection cassette (e.g., Hyg, Neo, etc.) flanked by I-CeuI and PI-SceIrestriction enzyme sites, and (2) inserting the TdT construct into themouse IgK BAC by I-CeuI and PI-SceI ligation. The final LTVEC depictedin FIG. 2.

This final LTVEC contains, from 5′ to 3′: (1) a Spec cassette forselection in bacteria, (2) a 28591 bp 5′ mouse homology arm (GRCm38genome coordinates 6:70,725,823-70,754,415) containing the IgKc gene,the IgK 3′ enhancer, and the 3′ IgK recombining sequence (RS); the mousearm ends ˜2.6 kb 3′ of the RS, (3) PI-SceI site, (4) aloxp-UbCp-em7-hyg-loxp cassette for selection in ES cells or bacteria,(5) the construct described above in Example 1.1 and FIG. 1 containingthe Rag2 promoter-human TdTS and Rag1-EGFP genes, (6) I-CeuI site, (7) a44,900 bp 3′ mouse IgK homology arm (GRCm38 genome coordinates6:70,754,508-70,799,678), and (8) CM cassette for selection in bacteria.

The final LTVEC is depicted in FIG. 2, with the approximate positions ofvarious sequence junctions indicated in the figure. The junctions arealso summarized in Table 2 below.

TABLE 2 Sequence Junctions of Rag-TdT IgK LTVEC SEQ ID Junction NOSequence 1. (mouse   8 CATCCTTACATCTTTGTCATCCCCTGTATCAACA IgK/PI-Sce1/TGGAAAGGCATTAATG/ATCTATGTCGGGTGCGG loxp-Ub-HygAGAAAGAGGTAATGAAATGGCA/ACCGGTATAA cassette)CTTCGTATAATGTATGCTATACGAAGTTATATG CATGGCC 2. (loxp-  9TTCGTATAATGTATGCTATACGAAGTTATGTC Ub-Hyg GACCTCGAGGGGGGGCCC/ACCTCCAGCcassette/ TGCCTTACAGAAAAGCAAATGCTTGCTTGCA mouse Rag2) ACAATCACCT3. (mouse  10 TATTGCGTTTTTTTAATCCTTTCAGATAAAA Rag2/humanGACCTATTCACAATCAAAA/ATGGATCCACC TdTS) ACGAGCGTCCCACTTGAGCCCTCGGAAGAAGAGACCCC 4. (human 11 GCCCTGGCTGAGGGAAATTTTGGAACTCCCAG TdTS/AsiS1/GCTCCAGACCCATTCTTT/GCGATCGC/TTTAG mouse CAAAAGCCCCTCAGACTCAGGTATATTGCTCTRag2) CTGAATCTACTTT 5. (mouse 12 CCCAAAGGAAAACACATTGGCAAATACCAARag1/Fse1/ CTTCTATGTGGAGATCCTAT/GGCCGGCC/GG EGFP)GGATCCAGACATGATAAGATACATTGATGAG TTTGGACAAACCACAAC 6. (EGFP/ 13TCGACCAGGATGGGCACCACCCCGGTGAACA mouse GCTCCTCGCCCTTGCTCAC/CATGTTGGCTAARag1) GCTACCTGGGAACAATGGGGGGGGGGGGGGG AGTCAAG 7. (mouse  14ACCTCTGCTGTGTCTGCAAGTTTGGCTTGTTC Rag1/I-CeuI/CTGCTTCTGATTTTTGGG/TCTAGACCCCCGGG mouse IgK)CTCGATAACTATAACGGTCCTAAGGTAGCGA CTCGAG/CATAACCACTTTCCTGCTATGGATCTGTTAAATATCCGCCAAAGGCCAAG

The resulting LTVEC was linearized and electroporated into VELOCIMMUNE®ES cells that comprise a functional ectopic mouse Adam6 gene (see, e.g.,U.S. Pat. No. 8,642,835, incorporated herein by reference). Afterselection for Hyg-resistance, ES cells were screened by TAQMAN®modification of allele assay (Valenzuela et al, supra) to identifycorrectly targeted clones.

Example 1.3 Generation of Random Transgenic and Targeted ImmunoglobulinKappa Locus Insertion of Short Isoform of Human TdT (TdTS), Both UnderControl of Mouse Immunoglobulin Heavy Chain Intronic Enhancer (Eμ) andMouse IgV_(H)1-7 2 Promoter (mIgH-Eμ-V_(H)1-72-TdT tg andmIgH-Eμ-V_(H)1-72-TdT IgK, Respectively)

The same human TdTS gene as used to make the Rag-TdT in Examples 1.1 and1.2 (i.e., from ATG start codon to about 514 bp 3′ of the polyA signal),was placed under the control of the 689 bp mouse Eμ enhancer and 303 bpmouse IgV_(H)1-72 promoter. This construct was either randomlyintegrated into the mouse genome or targeted to the immunoglobulin K(IgK) locus. For targeted integration, the gene was inserted between thesame 5′ and 3′ mouse IgK homology arms as were used to make the LTVEC inExample 1.2.

Specifically, the final LTVEC contains, from 5′ to 3′ (FIG. 3): (1) aSpec cassette for selection in bacteria, (2) a 28591 bp 5′ mousehomology arm (GRCm38 genome coordinates 6:70,725,823-70,754,415)containing the IgK constant (IgKC) gene, the IgK 3′ enhancer, and the 3′IgK recombining sequence (RS); the mouse arm ends ˜2.6 kb 3′ of the RS,(3) I-CeuI site, (4) loxp-UbCp-em7-hyg-loxp cassette in reverseorientation for selection in ES cells or bacteria, (5) the same 34,573bp human TdTS gene used in Examples above in reverse orientation, (6)the 303 bp mouse IgHV1-72 promoter in reverse orientation (GRCm38 genomecoordinates 12:115,758,417-115,758,719), (7) The 689 bp mouse Eμenhancer (EcoRT-XbaI fragment, GRCm38 genome coordinates12:113,427,284-113,427,972) in reverse orientation, (8) PI-SceI site,(9) a 44,900 bp 3′ mouse IgK homology arm (GRCm38 genome coordinates6:70,754,508-70,799,678), and (10) CM cassette for selection inbacteria.

The approximate positions of the specific sequence junctions in thefinal vector are depicted in FIG. 3, and their sequences indicated inTable 3 below.

TABLE 3 Sequence Junctions of mIgH-Eμ-V_(H)1-72-TdT  IgK LTVEC SEQ IDJunction NO Sequence 1. (mouse/ 15 CATCCTTACATCTTTGTCATCCCCTGTAT ICeu1/CAACATGGAAAGGCATTAATG/TCGCTA loxp-Ub- CCTTAGGACCGTTATAGTTA/GGCCCCCCCTCGA HygGGTCGACATAACTTCGTATAGCATACATTATACGAAG cassette) 2. (loxp- 16GGCCATGCATATAACTTCGTATAGCAT Ub-Hyg ACATTATACGAAGTTATACCGGT/AAA cassette/GAATGGGTCTGGAGCCTGGGAGTTCCA human TdT) AAATTTCCCTCAGCCAGGGC 3. (human 17CGGGGTCTCTTCTTCCGAGGGCTCAAGT TdT/mouse  GGGACGCTCGTGGTGGATCCAT/GGTGAGIgHV1-72) GTCCTGTGTGCTCAGTAACTGTAAAGAGA ACAGTGATCTCATGT 4. (mouse  18TAGTTTCCCCAAACTTAAGTTTATCGACTTCTA Eμ/PI-AAATGTATTTAGAATTC/TGCCATTTCATTACC SceI/TCTTTCTCCGCACCCGACATAGATAAAGCTT/CA mouse IgK)TAACCACTTTCCTGCTATGGATCTGTTAAATAT CCGCCAAAGGCCAAG

The resulting LTVEC was linearized and electroporated into VELOCIMMUNE®ES cells that comprise a functional ectopic mouse Adam6 gene (see, e.g.,U.S. Pat. No. 8,642,835, incorporated herein by reference). Afterselection for Hyg-resistance, ES cell clones were screened by TAQMAN®for correct targeting to the mouse IgK locus (for mIgH-Eμ-V_(H)1-72-TdTIgK) or for transgene copy number (for mIgH-Eμ-V_(H)1-72-TdT tg).

Example 1.4 Generation of Targeted Immunoglobulin Kappa Locus Insertionas Well as Transgenic Human TdTS from TdTS cDNA

Alternatively, a TdTS cDNA is synthesized de novo (Blue Heron Bio) as a3682 bp DNA fragment and incorporated into a targeting vector forintroduction into ES cells. The targeting vector contains, from 5′ to3′, a PI-SceI site, the 689 bp mouse IgH intronic enhancer (EcoRI-XbaIfragment), the 303 bp mouse VH1-72 promoter, the 1530 bp CDS of humanTdTS (NCBI RefSeq NM_004088) with the 735 bp intron 2 retained betweenexons 2 and 3 for intron-mediated enhancement of expression, the 340 bphuman TdT 3′ UTR/polyA signal, NotI and SalI restriction enzyme sitesfor ligating in a loxp-neo-loxp cassette, and an I-CeuI site. The vectorwas inserted between the same 5′ and 3′ mouse IgK homology arms as wereused to make the LTVEC in Example 1.2 and either targeted to the IgKlocus or randomly integrated into the mouse genome.

The resulting LTVEC is linearized and electroporated into VELOCIMMUNE®ES cells that comprise a functional ectopic mouse Adam6 gene (see, e.g.,U.S. Pat. No. 8,642,835, incorporated herein by reference). Afterselection for Hyg-resistance, ES cell clones are screened by TAQMAN® forcorrect targeting to the mouse IgK locus (for IgK targeted version) orfor transgene copy number (for transgenic version).

Example 1.5 Mice Expressing Human TdTS

As described above, once correctly targeted ES cells are produced, theyare introduced into 8-cell stage (or earlier) mouse embryos by theVELOCIMOUSE® method, screened in a gain of allele assay, andsubsequently bred to homozygosity. Heterozygous or homozygous animalsexpress human TdTS as well as antibodies comprising human variable lightand heavy chain domains and mouse constant regions (as these micecomprise human immunoglobulin variable light and heavy gene segments atthe endogenous IgK and IgH loci, respectively: VELOCIMMUNE® mice).

Several versions of human TdTS mice were generated and tested, and thoseincluded random transgenic and IgK targeted TdTS both under the controlof Rag promoter and Eμ-V_(H)1-72 regulatory elements. Also included wereversions with one, two, or several copies of the TdTS transgene; as wellas versions generated from genomic TdT and cDNA TdT sequences. Theremaining examples demonstrate data obtained with mice comprising atransgene of Rag-genomic TdTS (tandem insertion of two copies onchromosome 1, as described in Example 1.1 above) andEμ-V_(H)1-72-genomic TdTS targeted to the IgK locus (Example 1.3).

First, mice are tested for expression of TdT. PT-PCR was used foramplify TdT transcripts from bone marrow of either VELOCIMMUNE® control,VELOCIMMUNE®+Rag-genomic TdTS transgene, orVELOCIMMUNE®+Eμ-V_(H)1-72-genomic TdTS targeted to the IgK locus mice.Total RNA was used for reverse transcription by SUPESCRIPT® III ReverseTranscriptase (Life Technologies) using Oligo-dT primer. PCR wasconducted using SsoAdvanced™ Universal SYBR® Green Supermix, withprimers for either Beta Actin (control), primers designed to amplifyexons 1-2, primers designed to amplify exons 4-6, primers designed toamplify exons 7-9, and primers designed to amplify exons 9-11. As shownin FIG. 4, presence of human TdT exons was detected in both versions ofthe mice, while absent from VELOCIMMUNE® control mice.

Example 2 Human Immunoglobulin Kappa Junctional Diversity in MiceComprising Human TdTS

To assess immunoglobulin repertoire sequence diversity in variousVELOCIMMUNE® human TdTS mouse models described above in Example 1, IgKsequences were amplified by 5′ RACE from spleens of various mice withmIgK constant primer and sequenced using Illumina MiSeq.

Specifically, splenic B cells were positively enriched from totalsplenocytes by magnetic cell sorting using anti-CD19 (mouse) magneticbeads and MACS® columns (Miltenyi Biotech). Total RNA was isolated fromthe purified splenic B cells using an RNeasy Plus RNA isolation kit(Qiagen) according to manufacturer's instructions. Reverse transcriptionwas performed to generate cDNA containing Igic constant region sequence,using a SMARTer™ RACE cDNA Amplification Kit (Clontech) and an Igicspecific primer (Table 4). During this process, a DNA sequence, which isreverse compliment to 3′ of primer PE2-PIIA, was attached to the 3′ endof the newly synthesized cDNAs. Purified Igic specific cDNAs were thenamplified by the 1st round PCR using the PE2-PIIA primer and an Igicconstant specific primer listed in Table 4. PCR products between 450-700bp were isolated using Pippin Prep (SAGE Science). These products werefurther amplified by a 2nd round PCR using primers listed in Table 4(“XXXXXX” represents a 6 bp index sequences to enable multiplexingsamples for sequencing). PCR products between 400 bp-700 bp wereisolated, purified, and quantified by qPCR using a KAPA LibraryQuantification Kit (KAPA Biosystems) before loading onto a Miseqsequencer (Illumina) for sequencing using Miseq Reagent Kits v3 (600cycles).

TABLE 4 Primers used in library preparation for Igk repertoire sequencing RT  IgK 5′-AAGAAGCACACGACTGAGGCAC-3′ primers(SEQ ID  NO: 19) 1^(st)   IgK  5′- round ConstantACACTCTTTCCCTACACGACGCTCTTCCGATCT PCR (SEQ ID GGAAGATGGATACAGTTGGTGC-3′primers NO: 20) PE2-PIIA 5′- (SEQ ID  GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNO: 21) AAGCAGTGGTATCAACGCAGAGT-3′ 2^(nd)   Forward 5′- round (SEQ ID AATGATACGGCGACCACCGAGATCTACACXXXX PCR NO: 22) XX PrimersACACTCTTTCCCTACACGACGCTCTTCCGATCT- 3′ Reverse5′-CAAGCAGAAGACGGCATACGAGATXXXXXX (SEQ ID GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT- NO: 23) 3′

For bioinformatics analysis, raw Illumina sequences were de-muliplexedand filtered based on quality, length and perfect match to kappaconstant region primer. Overlapping paired-end reads were merged andanalyzed using custom in-house pipeline. The pipeline used localinstallation of IgBLAST (NCBI, v2.2.25+) to align rearranged light chainsequences to human germline V and J gene database. Rearrangements wereconsidered productive if no stop codons were detected and VJ junctionwas in-frame with J segment. Otherwise rearrangements were considerednonproductive and excluded from analysis.

CDR3 sequences were extracted using International ImmunogeneticsInformation System (IMGT) boundaries. Junctional region betweenannotated V and J segments was classified as P and N nucleotides. Regionwith N/P additions was extracted from each sequence and its lengthcalculated. Diversity of antibody repertoire was calculated by analyzingunique clonotypes. Sequence diversity was defined as a number of uniqueCDR3 sequences in randomly chosen 10,000 reads.

FIG. 5 shows that up to 2-fold increase in the number of unique CDR3amino acid sequences was detected in human TdTS mouse models comparedwith VELOCIMMUNE® mice that did not comprise human TdTS. Increased CDR3diversity was also observed on the nucleotide level (data not shown).FIG. 5 only shows data obtained with mice comprising two copies oftransgene of Rag-genomic TdTS (Rag TdT Tg) and Eμ-V_(H)1-72-genomic TdTStargeted to the IgK locus (mIgH-Eμ-V_(H)1-72 TdT IgK) (both homozygousand heterozygous versions), while similar data was obtained from otherversions of the mice (not shown).

Example 3 Increase in Non-Germline Additions in Mice Comprising HumanTdTS

Percentage of non-germline nucleotide additions in CDR3 (which consistsof parts of both Vκ and Jκ Gene segments) was also determined from NextGeneration Sequencing described in Example 2 above.

As depicted in FIG. 6, about 45% of humanized kappa light chain in Bcells were shown to have non-germline additions in both versions ofhumanized TdTS mice as compared to about 10% in VELOCIMMUNE® mice thatcomprise a functional ectopic mouse Adam6 gene. Sequence analysis ofimmunoglobulin light chains from spleen showed 0 to 8 non-templateadditions in light chains of human TdTS mice (8 in the figure includessequences with 8 or more non-template additions).

Example 4 Human Light Chain CDR3 Lengths in Immunoglobulins Obtainedfrom Human TdTS Mice

CDR3 sequences were extracted using International ImmunogeneticsInformation System (IMGT) boundaries. Non-template nucleotides weredetermined based on known light chain V and J sequences.

As depicted in FIG. 7A, increased non-template additions observed in thetwo versions of the human TdTS mice described in Examples 2 and 3 aboveled to increase in kappa light chain CDR3 length compared to control(VELOCIMMUNE® mice that comprise a functional ectopic mouse Adam6 gene).As depicted in FIG. 7B, sequence analysis revealed no extensiveexonuclease activity affecting 5′ J trimming rates in Rag-TdTS mice(only data for heterozygous mice is depicted here) as compared tocontrol (VELOCIMMUNE® mice that comprise a functional ectopic mouseAdam6 gene).

Example 5 Human Light Chain Vκ and Jκ Gene Segment Usage in Human TdTSMice

As depicted in FIGS. 8A and 8B, introduction of human TdTS in the twoversions of the mice described un Examples 2 and 3 above did notsignificantly alter the usage of either Vκ gene segments or Jκ genesegments compared to VELOCIMMUNE® mice that comprise a functionalectopic mouse Adam6 gene.

Example 6 Junctional Diversity at Light Chain Lambda ImmunoglobulinLocus and Other Rearranging Loci in Mice Comprising Human TdTS

In addition to human immunoglobulin kappa locus, antigen-receptordiversity in other loci from B (λ light chain, heavy chain) and T (α/β)lymphocytes can be investigated.

For example, when C λ1-containing lambda light junctional diversity inVELOCIMMUNE® mice that comprise a functional ectopic mouse Adam6 genewas compared to the same VELOCIMMUNE® mice also comprising human TdTtransgenes described in Example 1 above using the same sequencing methodas described in Example 2 and the primers listed in Table 5, increasedsequence diversity (about 2 fold) was observed at the mouse lambda locusof transgenic mice (FIG. 9). In addition, we observed increased rate ofmouse immunoglobulin lambda non-template additions (FIG. 10). CDR3lengths of the lambda chains in the TdT transgenic mice are depicted inFIG. 11. Finally, no difference in the mouse V lambda usage was observedbetween the various tested animals (FIG. 12).

TABLE 5 Primers used in library preparation for IgL-C1 repertoire sequencing RT  IgL 5′-CACCAGTGTGGCCTTGTTAGTCTC-3′ primers(SEQ ID  NO: 24) 1^(st)  IgL  5′- round  Constant ACACTCTTTCCCTACACGACGCTCTTCCGATCT PCR (SEQ IDAAGGTGGAAACAGGGTGACTGATG-3′ primers NO: 25) PE2-PIIA 5′- (SEQ ID GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC NO: 21) TAAGCAGTGGTATCAACGCAGAGT-3′2^(nd)   Forward 5′- round (SEQ ID  AATGATACGGCGACCACCGAGATCTACACXXXXPCR NO: 22) XXACACTCTTTCCCTACACGACGCTCTTCCGAT Primers CT-3′ Reverse5′-CAAGCAGAAGACGGCATACGAGATXXXXXX (SEQ ID GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC NO: 23) T-3′

Additionally, to the VELOCIMMUNE® mice that comprise unrearranged humanheavy and light chain variable gene segments, including those comprisingfunctional ectopic mouse Adam6 gene as described above (e.g., U.S. Pat.Nos. 8,878,001; 9,078,418; 9,125,386, incorporated herein by reference),or mice comprising only unrearranged human heavy chain variable genesegments or unrearranged human light chain variable gene segments, otheranimals can be generated that contain a human TdTS. Some such animalsinclude those comprising a human lambda variable region either onendogenous mouse lambda or kappa locus (U.S. Pat. Nos. 9,035,128;9,066,502; 9,163,092; 9,120,662; 9,029,628; 9,006,511; 9,012,717), ahuman kappa variable region at the endogenous heavy chain locus (e.g.,U.S. Patent Application Publication No. 2012/0096572), a humanized TCRalpha and beta loci (e.g., U.S. Pat. No. 9,113,616) and variouspermutations thereof, dual light chain mice and permutations thereof (USPatent Application Publication No. 2013/0198880), universal light chainmice and permutations thereof (e.g., US Patent Application PublicationNos. 2011/0195454; 2013/018582), universal heavy chain mice andpermutations thereof (e.g., U.S. Pat. No. 9,204,624), mice comprisinghistidine substitutions in their germline genome (e.g., U.S. Pat. Nos.9,334,334 and 9,301,510, US Patent Application Publication Nos.2013/0247236, 2014/0013456), chimeric antigen receptor mice (e.g., USPatent Application Publication No. 2016/0081314), mice lacking a CH1domain (e.g., U.S. Pat. No. 8,754,287 and US Patent ApplicationPublication No. 2015/0289489), all incorporated herein by reference. Anysuch animals where one desires to increase junctional diversity eitherat the light and/or heavy chain (e.g., human light and/or heavy chain)may be generated by introducing into ES cells comprising suchmodifications either a transgene or a targeted insertion of human TdTSdescribed herein. In case of mice generated from ES cells comprisingrandomly integrated TdTS transgene (and in cases where IgK locus has notbeen modified, e.g., humanized TCR loci mice), they can be alsogenerated by breeding with mice comprising various above-mentionedmodifications. Successful incorporation of TdTS allele into such animalsis determined as described herein above, and effect of human TdTSexpression on generation of junctional diversity at various loci isdetermined as described herein above. The effect on non-modifiedrearranging loci, e.g., endogenous mouse immunoglobulin and T cell loci,is also studied.

One such example, where the effect of TdTs introduction on junctionaldiversity of Dual Light Chain mice was studied, is presented in Examples7-10 below.

Example 7 Human Immunoglobulin Kappa Junctional Diversity in Dual LightChain (DLC) Mice Comprising Human TdTS

Mice comprising a dual light chain locus and human TdTS were generatedby breeding VELOCIMMUNE® mice comprising a functional mouse Adam6 gene(see U.S. Pat. Nos. 8,642,835 and 8,697,940) and exogenous human TdTSwith mice comprising the dual light chain locus (see U.S. PatentApplication Publication Number US 2013/0198880, incorporated herein byreference).

To assess immunoglobulin repertoire sequence diversity in DLC human TdTSmouse models that have a limited IgK loci containing only twounrearranged VK gene segments: IGVK3-20 and IGVK1-39, and fiveunrearranged IGJK gene segments (see U.S. Patent Application PublicationNumber US2013/0198880, incorporated herein by reference), IgK sequenceswere amplified by 5′ RACE from spleens of various mice with mIgKconstant primer and sequenced using Illumina MiSeq. In most experiments,several mice heterozygous for Rag TdT Tg and homozygous for the DLClocus (Rag TdT tg (HET) DLC) and two mice homozygous for Rag TdT Tg andhomozygous for the DLC locus (Rag TdT tg (HO) DLC) were used; data forRag TdT tg (HET) DLC is depicted as mean of all mice tested, while thetwo Rag TdT tg (HO) DLC mice are shown individually.

Specifically, splenic B cells were positively enriched from totalsplenocytes by magnetic cell sorting using anti-CD19 (mouse) magneticbeads and MACS® columns (Miltenyi Biotech). Total RNA was isolated fromthe purified splenic B cells using an RNeasy Plus RNA isolation kit(Qiagen) according to manufacturer's instructions. Reverse transcriptionwas performed to generate cDNA containing Igic constant region sequence,using a SMARTer™ RACE cDNA Amplification Kit (Clontech) and an Igicspecific primer (Table 4). During this process, a DNA sequence, which isreverse compliment to 3′ of primer PE2-PIIA, was attached to the 3′ endof the newly synthesized cDNAs. Purified Igic specific cDNAs were thenamplified by the 1st round PCR using the PE2-PIIA primer and an Igicconstant specific primer listed in Table 4. PCR products between 450-700bp were isolated using Pippin Prep (SAGE Science). These products werefurther amplified by a 2nd round PCR using primers listed in Table 4(“XXXXXX” represents a 6 bp index sequences to enable multiplexingsamples for sequencing). PCR products between 400 bp-700 bp wereisolated, purified, and quantified by qPCR using a KAPA LibraryQuantification Kit (KAPA Biosystems) before loading onto a Miseqsequencer (Illumina) for sequencing using Miseq Reagent Kits v3 (600cycles).

For bioinformatics analysis, raw Illumina sequences were de-muliplexedand filtered based on quality, length and perfect match to kappaconstant region primer. Overlapping paired-end reads were merged andanalyzed using custom in-house pipeline. The pipeline used localinstallation of IgBLAST (NCBI, v2.2.25+) to align rearranged light chainsequences to human germline V and J gene database. Rearrangements wereconsidered productive if no stop codons were detected and VJ junctionwas in-frame with J segment. Otherwise rearrangements were considerednonproductive and excluded from analysis.

CDR3 sequences were extracted using International ImmunogeneticsInformation System (IMGT) boundaries. Junctional region betweenannotated V and J segments was classified as P and N nucleotides(non-template additions). Region with N/P additions was extracted fromeach sequence and its length calculated. Diversity of antibodyrepertoire was calculated by analyzing unique clonotypes. Sequencediversity was defined as a number of unique CDR3 sequences in randomlychosen 10,000 reads.

FIG. 13 shows that over a 2-fold increase in the number of unique CDR3amino acid sequences was detected in DLC human TdTS mouse modelscompared with DLC mice that did not comprise introduced human TdTS.

Example 8 Increase in Non-Germline Additions in DLC Mice ComprisingHuman TdTS

Percentage of non-germline nucleotide additions in CDR3 (which consistsof parts of both Vκ and Jκ gene segments) in immunoglobulin sequences ofDLC TdT mice was also determined from Next Generation Sequencingdescribed in Example 7 above.

As depicted in FIG. 14, about half of humanized kappa light chains in Bcells were shown to have non-germline additions in DLC humanized TdTSmice (both HET and HO for TdT) as compared to about 10% in DLC controlmice (DLC mice with no introduced human TdT).

Example 9 Human Light Chain CDR3 Lengths in Immunoglobulins Obtainedfrom DLC Mice Comprising Human TdTS

CDR3 sequences were extracted using International ImmunogeneticsInformation System (IMGT) boundaries. Non-template nucleotides weredetermined from known light chain V and J sequences.

As depicted in FIG. 15, increased non-template additions observed in theDLC humanized TdTS mice (both HET and HO) described led to increase inkappa light chain CDR3 length compared to control (DLC mice with nointroduced human TdT).

Example 10 Human Light Chain Vκ and Jκ Gene Segment Usage in DLC MiceComprising Human TdTS

As depicted in FIG. 16, introduction of human TdTS in DLC mice did notsignificantly alter the usage of either Vκ gene segments or Jκ genesegments compared to DLC control mice (DLC mice with no introduced humanTdT).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A genetically modified mouse comprising in itsgermline genome: a nucleic acid sequence encoding a human TerminalDeoxynucleotidyltransferase (TdT) operably linked to a human or mousetranscriptional control element, wherein the transcriptional controlelement is active in pre-B cells; a human immunoglobulin heavy chainvariable region comprising unrearranged human immunoglobulin heavy chainV, D, and J gene segments operably linked to a human or mouseimmunoglobulin heavy chain constant region; and a human immunoglobulinlight chain variable region comprising unrearranged human immunoglobulinlight chain V and J gene segments operably linked to a human or mouseimmunoglobulin light chain constant region, wherein productiveantibodies of the genetically modified mouse have at least 10% greaterfrequency of unique immunoglobulin light chain CDR3 sequences thanproductive antibodies of a corresponding mouse that lacks human TdT. 2.The genetically modified mouse of claim 1, wherein the nucleic acidsequence encoding the human TdT is located at an immunoglobulin κ lightchain locus, an immunoglobulin λ light chain locus, an immunoglobulinheavy chain locus, a RAG1 locus, or a RAG2 locus.
 3. The geneticallymodified mouse of claim 1, wherein at least 20% of V-J immunoglobulinlight chain junctions in the mouse comprise non-template additions. 4.The genetically modified mouse of claim 1, wherein the immunoglobulinlight chain constant region is a mouse immunoglobulin light chainconstant region.
 5. The genetically modified mouse of claim 4, whereinthe immunoglobulin variable region and the mouse immunoglobulin lightchain constant region are located at an endogenous immunoglobulin lightchain locus.
 6. The genetically modified mouse of claim 1, wherein theimmunoglobulin heavy chain constant region is mouse immunoglobulin heavychain constant region.
 7. The genetically modified mouse of claim 6,wherein human immunoglobulin heavy chain variable region and theimmunoglobulin heavy chain constant region are located at an endogenousimmunoglobulin heavy chain locus.
 8. The genetically modified mouse ofclaim 1, wherein the human immunoglobulin light chain V and J genesegments are human Vκ and Jκ gene segments.
 9. The genetically modifiedmouse of claim 1, wherein the human immunoglobulin light chain V and Jgene segments are human Vλ and Jλ gene segments.
 10. The geneticallymodified mouse of claim 8, wherein the immunoglobulin light chainconstant region is a κ constant region.
 11. The genetically modifiedmouse of claim 9, wherein the immunoglobulin light chain constant regionis a λ constant region.
 12. The genetically modified mouse of claim 10,wherein the κ constant region is a mouse κ constant region.
 13. Thegenetically modified mouse of claim 11, wherein the λ constant region isa mouse λ constant region.
 14. The genetically modified mouse of claim12, wherein the immunoglobulin variable region and the mouse κ constantregion are located at an endogenous immunoglobulin κ locus.
 15. Thegenetically modified mouse of claim 13, wherein the immunoglobulinvariable region and the mouse λ constant region are located at anendogenous immunoglobulin λ locus.
 16. The genetically modified mouse ofclaim 1, wherein the TdT is a short isoform of human TdT (TdTS).
 17. Thegenetically modified mouse of claim 1, wherein the human or mousetranscriptional control element is a RAG1 transcriptional controlelement, a RAG2 transcriptional control element, an immunoglobulin heavychain transcriptional control element, an immunoglobulin κ light chaintranscriptional control element or an immunoglobulin λ light chaintranscriptional control element.
 18. A genetically modified mousecomprising in its germline genome: a nucleic acid sequence encoding ahuman Terminal Deoxynucleotidyltransferase (TdT) operably linked to ahuman or mouse transcriptional control element, wherein thetranscriptional control element is active in pre-B cells; a humanimmunoglobulin heavy chain variable region comprising unrearranged humanimmunoglobulin heavy chain V, D, and J gene segments operably linked toa human or mouse immunoglobulin heavy chain constant region; and a humanimmunoglobulin light chain variable region comprising unrearranged humanimmunoglobulin light chain V and J gene segments operably linked to ahuman or mouse immunoglobulin light chain constant region, wherein thenucleic acid sequence encoding the human TdT is located at animmunoglobulin κ light chain locus, and wherein the human or mousetranscriptional control element is a mouse RAG1 transcriptional controlelement, a mouse RAG2 transcriptional control element, a mouseimmunoglobulin heavy chain transcriptional control element, a mouseimmunoglobulin κ light chain transcriptional control element, or a mouseimmunoglobulin λ light chain transcriptional control element.
 19. Thegenetically modified mouse of claim 1, wherein the nucleic acid sequenceencoding the human TdT was randomly inserted into the genome, andwherein the human or rodent transcriptional control element is a mouseRAG1 transcriptional control element, a mouse RAG2 transcriptionalcontrol element, a mouse immunoglobulin heavy chain transcriptionalcontrol element, a mouse immunoglobulin κ light chain transcriptionalcontrol element, or a mouse immunoglobulin λ light chain transcriptionalcontrol element.
 20. A genetically modified mouse comprising in itsgermline genome: a nucleic acid sequence encoding a human TerminalDeoxynucleotidyltransferase (TdT) operably linked to a human or mousetranscriptional control element, wherein the transcriptional controlelement is active in pre-B cells; a human immunoglobulin heavy chainvariable region comprising unrearranged human immunoglobulin heavy chainV, D, and J gene segments operably linked to a human or mouseimmunoglobulin heavy chain constant region; and a human immunoglobulinlight chain variable region comprising unrearranged human immunoglobulinlight chain V and J gene segments operably linked to a human or mouseimmunoglobulin light chain constant region, wherein: (a) the human ormouse transcriptional control element is a mouse RAG2 transcriptionalcontrol element, and the nucleic acid sequence was randomly integratedinto the germline genome without disrupting any endogenous codingregions; (b) the human or mouse transcriptional control element is amouse RAG2 transcriptional control element, and the nucleic acidsequence is located at an immunoglobulin κ light chain locus; or (c) thehuman or mouse transcriptional control element is a mousetranscriptional control element comprising a mouse IgVH1-72 promoter anda mouse Eμ enhancer, and the nucleic acid sequence was randomlyintegrated into the germline genome without disrupting any endogenouscoding regions; or (d) the human or mouse transcriptional controlelement is a mouse transcriptional control element comprising a mouseIgVH1-72 promoter and a mouse Eμ enhancer, and the nucleic acid sequenceis located at an immunoglobulin κ light chain locus.
 21. The geneticallymodified mouse of claim 8, wherein at least 20% of V-J immunoglobulin κlight chain junctions in the mouse comprise non-template additions.